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I ’0‘ 'x\.u.‘(k . :22 Aug (v 1%" ' ‘5 M," ‘m: 552.:TE’TML 9f ‘ “(”37“ .giiu'f“ “‘3‘“;"”O an}. . ‘ " "-{V-dj‘d‘ r: ‘ "W3; ”A .135. a. 0:, 0 \‘l-p H >-. - -d..4-L .. 323% 4 - v 4 '3in f «mum 1: lmst’k..'}\ ‘— "1“‘H' w .4 AL- , all”; .. ,. , fi.fi3 v.‘ by ,I. . u. ‘ ,. . C v p.‘ 2.,1 ‘ 1 ¥ ' .2 :‘I’RJJ, 1‘”; p - 1'. ; .L f. r' "2 .3 ’0" ‘. N'- >~‘I a .4 :4! rg't.“w“ {mtg-N. . , 41%;» 4» 4. .., . ”1., .4 @1726me “5‘3 a'.’\\. ..,fi .. . J .2. ’ 9' . h“ ,. . - "(£23. -""~ "I A4‘ )3. —.. ,{.9 '2 .’ 2.1“ J MICHIGAN TATE I Ill/Hill:lllllll’l ’8' l" l 3 1293 0089 ll m nits ARIES l 1: III/Ill“ ml I! 5 7 97 This is to certify that the dissertation entitled Molecular Cloning, cDNA Sequencing, Expression in E. Coli, Active Site Identification and Catalytic Mechanism of Pig Liver Thioltransferase presented by Yanfeng Yang has been accepted towards fulfillment of the requirements for Ph.D. Biochemistry degree in 4242212444 Major professor Date A; £f/70 MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 MOLECULAR CLONING, cDNA SEQUENCING, EXPRESSION IN E, COLI, ACTIVE SITE IDENTIFICATION AND CATALYTIC MECHANISM OF PIG LIVER THIOLTRANSPERASE by Yanfeng Yang A DISSERTATION Submitted to Michigan State University in partial fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY 'Department of Biochemistry 1990 Tr. enzyme 1? Heights 1 Studies 3 important enzyme a; thiol-dis‘ consists CFSteine- A t 1“" cLi‘.‘ _ | For Verif oligonUr] of PUT. 5%“ sir The amine With that l acetllala rt) .. E8"- 2297:; ABSTRACT MOLECULAR CLONING, cDNA SEQUENCINC, EXPRESSION IN E. C L_, ACTIVE SITE IDENTIFICATION AND CATALYTIC MECHANISM OF PIG LIVER THIOLTRANSFERASE by Yanfeng Yang Thioltransferase, also known as glutaredoxin, is a cytosolic enzyme that catalyzes the reduction of disulfides of various molecular weights in the presence of a monothiol, especially glutathione (GSH). Studies have led to the suggestion that thioltransferase may play an important role in the regulation of protein function, for example, enzyme activity, by altering the thiol or disulfide status through thiol-disulfide exchange reactions. Pig liver thioltransferase (PLTT) consists of 105 amino acids with a molecular weight of 11,740, and cysteine-22 has been proposed to be the active site of the enzyme. A thioltransferase cDNA clone was obtained by screening a pig liver cDNA library in A-gtll with polyclonal antibodies against PLTT. For verification, this isolated cDNA was hybridized with three oligonucleotides synthesized according to the known amino acid sequence of PLTT. The confirmed PLTT cDNA was subcloned into Ml3mp18 at the EQQRI site and sequenced using the dideoxy chain—termination method. The amino acid sequence deduced from the cDNA sequence agreed exactly with that determined directly, except that the N-terminus should be N- acetylalanine followed by glutamine rather than the reverse as originally reported. This CDNA, with an introduced Ecol site at its initiation codon region, was cloned into an expression vector, pKK233~2, between the NcoI and HindIII sites and expressed in E. coli JM105 at a high'le ijei'if J l . ..,. JibU;.l high-level (8% of total soluble protein). The recombinant enzyme was identical to the native enzyme in amino acid composition, thiol- disulfide exchange activity, and kinetic properties except no N- acetylation occurred at its N-terminus (alanine). The speculated active site, Cysn-Pron-Pheu-Cyszs-Arg26-LysN, of 22 22 25 25 PLTT was directly tested by exchanging Cys with Ser , Cys with Ser or Ala”, Arg26 with Val26 and Lys27 with Glnn. Comparison of these H mutants with the wild-type enzyme revealed that Cys is the catalytic site and its low pKi (3.8) is facilitated by Argu. Not expected was the M finding that the Set mutant had an increased rather than decreased activity, indicating that the formation of an intramolecular disulfide 22 25 is not required for the enzyme catalytic H between Cys and Cys mechanism of mutants at position 25. However, the Ala mutant lost 91% of the wild-type enzyme activity, suggesting that an amino acid with a more polar side chain than a methyl group, such as -CH20H or -CHZSH, is 27 required at position 25. The roles of Lys and the second pair of n M, were also investigated. Alternative cysteines, Cys and Cys catalytic mechanisms for thioltransferase were proposed according to radioactive labeling and kinetic studies of these mutant enzymes. Chapter II was published in Gene, volume 83, pp 339-346 (1989). Chapter III was published in the Journal of Biological Chemistry, volume 265, pp 589-593 (1990). This work is reprinted here by permission of the publishers. Chapters IV, V, and VI were written in formats suitable for publication and submitted to the Journal of Biological Chemistry. To my mother and father as well as my wife and son DESI: EHCOL as a labs: CIOn Dan. My Hr ACKNOWLEDGMENTS I wish to express my special gratitude to my major professor and mentor Dr. William W. Wells for his intellectual instruction, cordial encouragement and financial support. I have really enjoyed these years as a graduate student in his laboratory. I would also like to thank those professors who have served on my guidance committee, Drs. Zachary Burton, Susan E. Conrad, Thomas Deits, and Robert Hausinger for their special advice and invaluable time contributed to my research. I also owe thanks to the former and present members of this laboratory, Dr. Zhong-Ru Gan, Pamela Rocque, Dr. Hiroyuki Arai, Dian Peng Xu, and Keri Gardner for their friendship and help. I also acknowledge Dr. Arnold Revzin and the people in his laboratory for offering their assistance and experience for my molecular cloning work. Finally, my deepest thanks is for my wife, Jianli, and my son, Dan, whose love and full cooperation are always the spiritual source of my working energy. vi LIST 0! LIST Ol LIST Of CHAPTEE Prote Disul Thior Thiol REFER mm; SLXVA 1m TABLE OF CONTENTS LIST OF TABLES x LIST OF FIGURES xi LIST OF ABBREVIATIONS xiii CHAPTER I. LITERATURE REVIEW 1 Protein-Disulfide Isomerase and Thiol:Protein Disulfide Oxidoreductase 4 Thioredoxin 8 Thioltransferase (Glutaredoxin) 13 REFERENCES 22 CHAPTER II. CLONING AND SEQUENCING THE cDNA ENCODING PIG LIVER THIOLTRANSFERASE 32 SUMMARY 33 INTRODUCTION 34 MATERIALS AND METHODS 36 RESULTS AND DISCUSSION 42 Antibody Screening of Thioltransferase cDNA 42 Oligonucleotide-probe Hybridizations 42 Western-blotting Analysis of the Fusion Protein 43 Nucleotide Sequence Analysis 44 REFERENCES 59 CHAPTER III HIGH-LEVEL EXPRESSION OF PIG LIVER THIOLTRANSFERASE (GLUTAREDOXIN) IN ESCHERICHIA COLI 62 vii Ex; Ami Seq Iso RIFEEI CHAPTER 1 EXpr Thic D I‘m.“ PEFEF; CHAPTER SUMMARY — ————— — ————————— 63 INTRODUCTION ------------------------------------ 64 EXPERIMENTAL PROCEDURES -- 66 RESULTS ---------------------------------------- 70 Construction of PLTT Expression Vector 70 Expression and Purification of PLTT ---- 7O Amino Acid Composition and N-terminal Sequence Analysis — 75 Isoelectric Focusing .75 Optimum pH and Kinetic Behavior - 80 DISCUSSION 86 REFERENCES — — -------- — 89 CHAPTER IV. IDENTIFICATION OF THE FUNCTIONAL AMINO ACIDS AT THE ACTIVE CENTER OF PIG LIVER THIOLTRANSFERASE BY SITE—DIRECTED MUTAGENESIS 91 SUMMARY 92 INTRODUCTION 94 EXPERIMENTAL PROCEDURES 96 RESULTS ~-- 100 Mutagenic Oligonucleotides and Site-Directed Mutagenesis --- 100 Expression and Purification of Mutant Thioltransferase ----- 102 Thioltransferase Activity Comparison — — 105 DISCUSSION * - 113 REFERENCES --- 119 CHAPTER V. CHARACTERIZATION OF MUTANT PIG LIVER THIOLTRANSFERASE - -- ------- 122 SUMMARY ------------- 123 INTRODUCTION ---------------------------------------------- 125 EXPERIMENTAL PROCEDURES ~~~~~ - 127 viii o- «"v p .h~. ._ ‘ .-< '1'! ‘w-o--, LIFE??? Er-v 'v w ~VL .. . RESULTS AND DISCUSSION ——————————————————————————————————————— 13o Isoelectric Focusing Analysis ------------------------------ 130 Optimum pH and Kinetic Behavior ---------------------------- 134 The pKi‘Values of the Expressed Thioltransferases ---------- 139 The DHA Reductase Activity rr-r 146 REFERENCES ------------------ —-- ----------- 150 CHAPTER VI. EVIDENCES FOR THE CATALYTIC MECHANISM OF THIOLTRANSFERASE --- 152 SUMMARY ------- 153 INTRODUCTION - — — - 154 EXPERIMENTAL PROCEDURES — r ------ 157 RESULTS -- - - 160 Preincubation and Inhibition Studies 160 Isoelectric Focusing Analysis — 169 Radioactive Labeling Studies 172 DISCUSSION 177 REFERENCES -—- r - 180 CONCLUSION ---------------------- —- --------------- 182 ix Table I. 1 II. c 2 III. P N. A r V o In: n. p. :1 VII. R LIST OF TABLES Table I. Enzyme activity modified by thiol-disulfide exchange -------- II. Oligonucleotide probes for hybridization of cDNA for pig liver thioltransferase III. Purification of recombinant pig liver thioltransferase ------ IV} Andno acid composition comparison of purified recombinant and native pig liver thioltransferases V. Oligonucleotide primers used in site-directed mutagenesis of pig liver thioltransferase VI. Purification of wild-type and mutant Page 38 76 79 101 thioltransferase by CM Sepharose chromatography 108 173 VII. Radioactive labeling of thioltransferases 10. 11. 12. 13. 15. 16, Nucl and of ; tab: Autc of t Cons SDS+ cell SDS— puri 1809 LIST OF FIGURES Figure l. The three dimensional structures of E. coli and T4 thioredoxins 2. Western-blot analysis of the fusion protein 3. Polyacrylamide gel electrophoresis isolation of pig liver thioltransferase cDNA 4. Pig liver thioltransferase cDNA sequencing strategy 5. Nucleotide sequence of pig liver thioltransferase and its deduced amino acid sequence 6. Comparison of the Nrterminal amino acid sequence of pig liver thioltransferase, calf thymus and rabbit born marrow glutaredoxins 7. Autoradiography of the 5'-end nucleotide sequence of the cDNA for pig liver thioltransferase 8. Construction of the PLTT expression vector pTTl 9. SDS-PAGE and immunoblotting analysis of the total cell proteins containing the expressed PLTT ‘— 10. SDS-PAGE analysis of various steps in the purification of expressed PLTT 11. Isoelectric focusing of PLTT 12. Comparison of the recombinant and native PLTT on optimum pH and kinetic behavior 13. Nucleotide sequencing of mutant cDNAs 14. SDS-PAGE and immunoblotting analysis of the expressed mutant pig liver thioltransferase in cell extracts 15. SDS-PAGE analysis of the purified mutant pig liver thioltransferases ————— - 16. Enzyme activity comparison of mutant and xi Page 10 46 48 51 53 56 58 72 74 78 82 84 104 107 110 17. 18. 19. 20. 21. 28. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. wild-type pig liver thioltransferases — Comparison of amino acid sequence in two conserved regions --- Isoelectric focusing of mutant thioltransferases Optimum pH of mutant thioltransferases Kinetic behavior of mutant thioltransferases A plot of the alkylation reaction between the samfi concentrations (60 uM) of reduced ETT-Ser2 and iodoacetamide pH dependence of second order apparent rate constant between each of the reduced thioltransferases (60 uM) and iodoacetamide (60 uM) Comparison of the DHA reductase activity of thioltransferases Inhibition of pig liver thioltransferases Differential preincubation of pig liver thioltransferases ---- Protection of pig liver thioltransferases by HED Isoelectric focusing analysis of the ES intermediates of thioltransferase Autoradiography of ETT and ETT-Ser25 xii 113 116 132 136 138 142 144 148 162 164 168 171 176 Cys-S: DEA ETT‘an B‘gal X-gal GIT HED 1AA 1AM IPTG Cys-SO{ DHA DMSO DTT ETT ETT-ami no ac id. B-sal X-gal GIT HED IAA IAM IPTG LB-medium PDI PLTT PMSF PPO RRase SDS-PAGE TBST LIST OF ABBREVIATIONS S-sulfocysteine dehydroascorbic acid dimethylsulfoxide dithiothreitol expressed pig liver thioltransferase expressed mutant thioltransferase with the mutant amino acid's position B-galactosidase 5-bromo-4-chloro-3-indolyl- ngfgalactopyranoside GSH-insulin transhydrogenase hydroxyethyl disulfide iodoacetic acid iodoacetamide isopropyl-Bfgrthiogalactopyranoside Luria-Bertani medium protein disulfide isomerase pig liver thioltransferase phenylmethylsulfonyl fluoride 2,5-diphenyloxazole ribonucleotide reductase sodium dodecyl sulfate polyacrylamide gel electrophoresis Tris buffered saline with 0.05% Tween-20 xiii CHAPTER I LITERATURE REVIEW equxl: cellul reduct activi enzyxw bisphc metabo treate the la are ca disulf Oxidat enzyme ( which ‘ de R..- and Und The 03H the Fat 'Y- It is well known in recent years that the thiol-disulfide equilibrium directly or indirectly exerts an influence on various cellular processes, such as enzyme regulation (1-14), ribonucleotide reduction (15,16), protein synthesis and modification (17-19), calcium metabolism (20), lipid peroxidation (21,22), and hormone metabolism (23,24). In most cases, if not all, the modulation of the above activities is fulfilled by modification of thiols or disulfide of the enzymes through reversible oxidoreduction. For example, fructose 1,6- bisphosphatase and phosphofructokinase, two key enzymes in carbohydrate metabolism, are regulated by the thiol/disulfide ratio, ip_vitro, when treated with the disulfide cystamine. The former is stimulated whereas the latter is inhibited (25). In principle, accessible enzyme thiols are capable of forming protein-mixed disulfide or intramolecular disulfide, i vivo, by reaction with cellular disulfides. Such oxidation could result in either increasing, decreasing or not affecting enzyme activity, some examples of each are listed in table I. Glutathione (£31-glutamyljéécysteiny1-glycine), a tripeptide, which was found and originally named "philothion" one century ago by J. de Rey-Pailhade (26), is the major cellular thiol/disulfide redox buffer and undergoes thiol (GSH)-disulfide (GSSG) interchanges, ip_yiyg (27). The GSH/GSSG ratio in cells is high with a range between 100 to 300, and the ratio may be changed by hormone induction, as reported (28). In such a strongly reduced environment, the ratio of spontaneous thiol/ disulfide exchange between protein thiols and low molecular-weight disulfide is considerably slow at physiological pH and is non-specific (29). Accordingly, there is likely to be a class of enzymes that Glyccg FhOSph Fructc HGXOR; clUCO: PFFUV “ ‘ Enzyme activity modified by disulfide treatment Table I Enzymes Sources effect References Glycogen phosphorylase rabbit liver inactivation l phosphatase Glycogen Synthase D rabbit liver inactivation 2 Phosphofructokinase rabbit skeletal inactivation 3 muscle Fructose 1,6- rabbit liver activation 4 bisphosphatase Hexokinase bovine brain inactivation 5 Glucose-6-phosphatase rabbit liver no effect 6 Pyruvate kinase rat liver inactivation 7 Glucose-6-phosphate rat liver, heart activation 9 dehydrogenase Acetyl-CoA hydrolase rat pineal activation 13 Acid phosphatase spinach activation 14 catalyze several 4 have bee: refold t nonenzyu; for a ce 4 catalyze these reactions ig vivo. In the past couple of decades, several enzymes with general thiol-disulfide interconversion activity have been investigated in detail. Protein-Disulfide Isomera§g_§nd Thiol:Protein Disulfide Oxidoreductase Reduced unfolded proteins can spontaneously reoxidize and refold to form their native functional disulfide conformation. The nonenzymatic process, however, is not efficient (30). During a search for a cellular catalyst for this process, protein disulfide isomerase (PDI, EC 5.3.4.1.) was independently identified by the research groups of Anfinsen (31) and Straub (32). PDI is thought to catalyze thiol/disulfide exchange reactions involved in the posttranslational formation of disulfide bonds. It also can rearrange the disulfide bonds of a denatured "scrambled" protein (e.g. ribonuclease) to the native form, and in the presence of reductant thiols, this reaction was used as an assay method for PDI activity (33). PDI is widely distributed in animal and plant tissues, especially rich in those cells with major roles in synthesis of disulfide-bond containing proteins, for instance, mammalian liver, pancreas, lymphoid, chick embryo, and wheat endosperm, which implies a correlation between the level of PDI and the extent of synthesis of secretory proteins (19). PDI is one of the most abundant proteins in microsomes and its subcellular location is found in the lumen of both rough and smooth endoplasmic reticulum and loosely associated with the surface of the ER membrane (34). The distribution and subcellular location of PDI are highly correlated with its speculated function, catalyzing the formation of disulfide bonds of newly synthesized proteins in the lumen of the endoplasmic reticulum. characteri amuse ay and an ac determine The dedu< and has ' the acti thioredc Contain CYS‘Lys disulfj Partiaz its d9< enl‘me to the the s; Prote 5 Since 19605, PDI has been purified to homogeneity and characterized from calf (35,36), rat (37,38), and mouse (39). This enzyme appears to be a homodimer with a molecular weight of 2 x 57,000 and an acidic pI of 4.2 (36). The primary structure of PDI was first determined by nucleotide sequencing of a cDNA for rat pancreas PDI (40) The deduced amino acid sequence consists of 508 amino acids (Mr 56,783) and has two internal homologous regions which are highly comparable to the active site sequence, Trp-Cys-Gly-Pro-Cys-Lys, of E, coli thioredoxin, a small cytosolic enzyme of 12 kDa. Both PDI regions contain the postulated active site with the sequence, Trp-Cys-Gly-His- Cys-Lys, confirming that it should have the ability to catalyze thiol- disulfide exchange reactions, a characteristic of thioredoxin (40). The partial sequence of a cDNA for human liver PDI was also determined and its deduced amino acid sequence showed 942 sequence homology to the rat enzyme, and like the rat enzyme, two sequences with similar identities to that of the E, £91; thioredoxin active site were found (41). When the sequences were reported, PDI was compared with numerous other proteins, and several extended roles for PDI were suggested. Prolyl-4-hydroxylase (EC 1.14.11.2), a tetrameric enzyme (aflfi) with a molecular weight of 250,000, catalyses the formation of 4-hydroxy proline in collagen and other proteins with collagen-like amino acid sequences by the hydroxylation of proline residues in peptide linkages (42). The B-subunit of this enzyme has been identified as PDI. Comparison of the cDNA sequences of the human hydroxylase B-subunit and that of rat PDI shows that the degree of homology was 84% and 94% at the level of nucleotide sequence and the deduced amino acid sequence, respectively. Southern blot analysis of human genomic DNA indicated only Cf isolate PDI i.s the the involve proteir binding to that A‘hydrc (#5). hydroxy and the Pathwa} l dESCrit PFOteir the N-] that of multipl Sequenc reticUl identic at thos Propobe miCFOSQI with I To: 6 only one gene containing the PDI sequence (43). In addition, the isolated B-subunit of prolyl-4-hydroxylase has PDI activity similar to PDI itself, and even in the intact tetramer form the subunit had 50% of the theoretical activity (43). These data imply that PDI might be involved in the synthesis of collagen. Another cDNA for human p55 protein, defined as thyroid hormone 3,3',S-triiodo-L-thyronine (T3) binding protein, was sequenced and its coding region was 85% homologous to that of rat PDI and 98% homologous to the B-subunit of human prolyl- 4-hydroxylase (44). The p55 protein was also shown to have PDI activity (45). It has been concluded that PDI, the B-subunit of prolyl-4- hydroxylase and the p55 protein are the products of the same gene (44), and that the PDI/B-subunit could also play a role in T3 transport pathways in the cell. In addition, there are some other proteins that are newly described as relatives of PDI. A chicken glycosylation site binding protein, a component of an oligosaccharyl transferase which catalyses the N-linked glycosylation on nascent proteins, shared 90% homology to that of rat PDI in amino acid sequence (46). It is likely that PDI has multiple functions in the modification of newly translated proteins. Sequencing of cDNA clones encoding two abundant lumenal endoplasmic reticulum proteins (ERp), ERp59 and ERp72, revealed that ERp59 is identical to PDI, and that ERp72 showed sequence homology with ERp59/PDI at those regions having copies of the sequence, Cys-Gly-His-Cys, the proposed active site of PDI (47). Another 58 kd protein of the microsomal triglyceride transfer protein complex (MTP) was recently identified as PDI by comparison of the properties of the two proteins with respect to N-terminal sequence, reverse phase HPLC maps, immunolo that all the lume and modi involved of prote answered effective Observe c define. s PDI (30). Physiolog regulated Pmteins Con Oxidoredu 1.8.4.2). of OSH, w. °FiginauJ yielded a of the her Purified f Similar to molecmar ‘ it was afgu FreQdman f’ g the stages ‘ 7 immunological reactivities, and enzyme activity (48). It is interesting that all these PDI-like proteins, as well as PDI itself, are located in the lumen of the endoplasmic reticulum where proteins are synthesized and modified. Thus, PDI is thought to be a multifunctional enzyme involved in cotranslational modifications and posttranslational folding of proteins (49). Questions about PDI function still remain to be answered, despite the present state of knowledge. PDI is not a very effective catalyst and rather high enzyme concentrations are needed to observe catalytic protein re-oxidation, in yitgg, and the lack of a well defined substrate has hampered the study of the catalytic mechanism for PDI (30). Studies, ip,yiyg, both on the catalytic reaction and on its physiological function are limited. How the multifunctional enzyme is regulated in playing role(s) either alone or associated with other proteins needs further investigation. Concurrently, a similar enzyme called thioltprotein disulfide oxidoreductase, also named glutathione-insulin transhydrogenase (GIT, EC 1.8.4.2), catalyzing thiol-disulfide exchange reactions in the presence of GSH, was studied by several groups (SO-57). This protein was originally isolated as an insulin disulfide reduction activity that yielded a trichloroacetic acid (TCA)-soluble insulin A chain by cleavage of the hormone's disulfide bond with GSH (50, 51). Since then GIT was purified from different species and tissues (50, 52-57). Because GIT is similar to PDI in nearly every respect, for example, in activity, molecular weight, pI, distribution in tissues and subcellular locations, it was argued in the 19705 that PDI and GIT were the same protein. Freedman, t l. corpurified the PDI activity and GIT activity through the stages of the purification from rat liver, and suggested that a single pr collabcra 11111511172511 im‘mo‘. ;;~ acceptei sane prc thiorec Cate} V enZFTne 8 single protein was responsible for both activities (58). The collaborative study of six preparations of GIT and PDI with double immunodiffusion and rocket-line immunoelectrophoresis confirmed the immunological identity of PDI and GIT (59). Today, it is generally accepted that PDI and GIT are identical, and represent two names for the same proteins. Recent observations from our laboratory revealed a further intrinsic function of PDI, namely dehydroascorbate reductase activity (60). Further discussion of this finding is given below. Thioredoxin In addition to PDI, three cytosolic low molecular weight proteins, thioredoxin, thioltransferase and glutaredoxin, have the ability to catalyze thiol-disulfide exchange reactions. Among these heat stable enzymes, thioredoxin is the most extensively studied (61, 62). In 1964, thioredoxin was first described as the hydrogen donor for E. ggli ribonucleotide reductase, an enzyme that catalyzes the formation of deoxyribonucleotides from the corresponding ribonucleotide (63). Since then, the ubiquitous protein has been purified from bacteriophage (64), bacteria (63, 65, 67), yeast (68, 69), green algae (70,71), plants (72-74), and animals (75-79). Thioredoxin from all organisms is a single polypeptide with 104-114 amino acids and a molecular weight of approximately 12,000, except that from bacteriophage (T4), which contains 87 amino acids and has no sequence homology and no immunological cross-reactivity with the E. ggli enzyme (77, 78). However, X-ray crystallography to 2.8 A resolution for both proteins showed that they share a similarity in folding and have common three dimensional structures with the active site disulfide bridge in a Fig. 1. The three dimensional structures of E. coli and T4 thioredoxins. Schematic backbone drawing of three dimensional structures of E. coli (left) and T4 (right) thioredoxins showing their similar folding. lO protrudin; thioredrx amino aci conserret sequence from a c I4 this: fact, 1' 97). thioreq disuli a gene Elect. thic tit: f<3r thic A rc)“ tha+ ll protruding loop (Fig. l) (82, 83). The primary structures of thioredoxin from different sources were determined either by direct amino acid sequence or deduced from their cDNA sequences with various conserved homologies among them, e.g., all contain the active center sequence, Cys-Gly-Pro-Cys (79, 84-95), implying all the proteins evolved from a common ancestor. The active site sequence, Cys-Val-Tyr-Cys, of T4 thioredoxin is more like that, Cys-Pro-Tyr-Cys, of glutaredoxin. In fact, the two proteins are closely similar in amino acid sequence (96, 97). The thioredoxin system, including thioredoxin, NADPH, and thioredoxin reductase, a FAD-containing protein with a redox-active disulfide, seems to participate in'many diverse biochemical processes as a general protein thiol-disulfide oxidoreductase by transferring electrons from NADPH to its substrate proteins as shown below (61): NADP‘ FADE2 TR-s2 X T-(SH)2 P—s2 Thioredox Reductase Thioredoxin Protein For E. ggli and mammalian cells, the biochemical function of thioredoxin was originally reported to be a hydrogen donor for ribonucleotide reductase, i3 yitgg, (64, 67). However, additional roles for the enzyme were later described. Pigiet and Schuster found that thioredoxin from E. ggli catalyzed the refolding of ribonuclease either from the reduced, denatured form or from the scrambled form, suggesting that thioredoxin may serve as a PDI analogue in E. goli (98). Rat liver thioredu maintair filamet synthe aggreg two 0 thior thior for . (87 , Plan elec 50m. 891'} in l2 thioredoxin could activate cytosol glucocorticoid receptor activity by maintaining the receptor in reduced form (99). T7 DNA polymerase posesses full activity only in the presence of E. ggli thioredoxin, indicating that thioredoxin is an essential subunit of T7 DNA polymerase (100). Thioredoxin also plays significant roles in the assembly of filamentous phages (fl, M13) (101) and in the initiation of protein synthesis (102). The process of reducing insulin disulfides to produce aggregating B chains is currently used to assay thioredoxin activity (103). Different from E. 9911 and mammalian cells, plant tissues contain two or more thioredoxin species (61). The best characterized plant thioredoxins are from spinach chloroplasts in which two types of thioredoxin exist, the f- and m- types, classified by their preference for enzyme activation (73). Both types have been purified and sequenced (87, 93), and show little sequence homology ((18%). In green tissues of plants, sunlight provides the energy for photosynthesis and, indirectly, electrons for the regulation of chloroplast enzyme activities (104). Some of the regulatory steps are connected by thioredoxins and the general pathway for thioredoxin-linked regulation is shown below PSIFC iXFTR- (SH)2 E- (SH)2 (active) Fdre FTR-82 T- (SH)2 E- 82 (inactive) in which PSI, Fd, FTR, T, and E represent photosystem I, ferredoxin, ferredoxin-thioredoxin reductase, thioredoxin, and target enzyme, are KW" palate thiorei malate 01 t1 13 respectively (105). The well-established examples of such regulation are two photosynthesis enzymes, fructose-l,6-bisphosphatase and NADPt- malate dehydrogenase which are specifically activated by f- and m- type thioredoxins, respectively. The f-type protein also works on NADPt- malate dehydrogenase but with different kinetics (73, 106). Thioredoxin was also shown to be essential for photosynthetic growth in cyanobacterium Anacysis nidulans R2, in vivo, since deletion of the gene of thioredoxin m is a lethal mutation (107). Recently, Holmgren's research group demonstrated that PDI from calf liver has intrinsic thioredoxin activity (catalysis of NADPH dependent insulin disulfide reduction) and is a substrate for thioredoxin reductase from calf thymus or rat liver (108). Thus, PDI might be a high molecular weight member of the thioredoxin family. Most observations on the functions of thioredoxin were obtained, iplyiggg, and the details of how this small enzyme functions in the above biochemical processes were not revealed. Hence, the mechanisms of the reactions and the true functions of thioredoxin in living cells require further studies. Thioltransferase (Glutaredoxin) Compared with thioredoxin, thioltransferase has a longer history. In 1955, Racker first discovered an enzyme with an activity that catalyzes the conversion of homocystine to homocysteine in the presence of GSH, GSSG reductase and NADPH, and named it GSH-homocystine transhydrogenase (109). In 1960, a protein with similar activity, but with preference to éfcystine as the substrate, was detected in yeast by Black's group (110), and several years later this protein was highly urified finding c glutatti- -‘ . 1. mJAELUA~3 “he :- e l4 purified and called thiol-disulfide transhydrogenase (111). With the finding of the native unsymmetrical disulfide of coenzyme A and glutathione, CoASSG, Chang and Wilken purified a enzyme, with a molecular weight of 12,000, from bovine kidney, which catalyzed the reduction of CoASSG to 00A and GSH and could not reactivate "scrambled" ribonuclease. They named the protein sulfhydryl-disulfide transhydrogenase (112). All the early reported enzymes catalyzing the GSH dependent reduction of low molecular weight disulfide substrates (reaction 1) were named "transhydrogenases" RSSR + 2GSH:2RSH + GSSG (1) where RSSR represents widely variable disulfide substrates. By examination of the mechanism of the above reaction (reaction 1), Mannervik and co-workers, who isolated a "transhydrogenase" from rat liver (113), pointed out that this thiol-disulfide exchange reaction catalyzed by the enzyme followed a thioltransferase mechanism via consecutive ionic displacement reactions RSSR + GSH:-_—_:t RSSG + RSH (2) R886 + GSH:————-CSSC + RSH (3) rather than the transhydrogenase mechanism, i.e., a one step three substrate reaction (114). The sum of reactions 2 and 3 equals reaction 1. The resulting GSSG can be reduced by GSSG reductase using NADPH as electron donor. They replaced the name "thiol (or GSH)-disulfide transhydrogenase" with "thioltransferase" which has been commonly accepted by most laboratories today. ' ‘ I j 1 low of ass Fri: (11‘ thi< aCIC 15 Characterization of viable E. 59;; mutants lacking detectable thioredoxin, but still maintaining full ribonucleotide reductase activity resulted in the discovery of glutaredoxin as a GSH-dependent hydrogen donor system including glutaredoxin, GSH, glutathione reductase and NADPH (115). Later, similarities in structure, catalytic activity, size, immunology and other properties between thioltransferase and glutaredoxin indicated that they were identical (see below). TT and GRX have been purified from E. EQEE (113,116), yeast (111,117), rat liver (113,118), calf thymus (119), bovine liver (120), human placenta (121), pig liver (122), and rabbit bone marrow (123). The molecular weight of thioltransferases or glutaredoxins from animal tissues and yeast is about 11,700, whereas that from E. ggli is gg, 10,000; all enzymes are located in the cytosol (124) and have catalytic activity toward various low molecular weight disulfides and protein disulfides in the presence of GSH, GSSG reductase, and NADPH. This reaction is used as a standard assay for thioltransferase thiol-disulfide exchange activity. The primary structures of E, 32;; (96, 125), calf thymus (126, 127), yeast (117), pig liver (128, 129), and rabbit bone marrow (123) thioltransferases (glutaredoxins) were determined either by direct amino acid sequence (96, 117, 125, 127) or deduced from their nucleotide sequence (125, 129). The three mammalian enzymes contain 105 amino acids (106 for rabbit protein) with two pairs of cysteines and share 85% sequence homology, whereas the yeast and E. 29;; enzymes contain 106 and 85 amino acids, respectively, with only two cysteine residues and have 32% sequence identity to each other. The sequence similarity between the pig and yeast protein is 51% and that between the calf and E._99EE polypeptide is 30%. However, all enzymes have the same active center If! ante hmmx live: thio cros EXF‘E pe r Kan at th 16 with the sequence of Cys-Pro-Tyr(Phe for pig enzyme)-Cys-, located in the N-terminal region, indicating they are probably from the same ancestral gene, especially regarding the mammalian proteins. Immunological studies showed that polyclonal antibodies against pig liver thioltransferase can recognize calf thymus glutaredoxin and calf liver thioltransferase with the same sensitivity, but not E, ggli thioredoxin (130). Antibodies against calf thymus glutaredoxin can cross-react with human placenta thioltransferase (131). The experimental data described above strongly support the conclusion that TT and GRX are two names for the same protein. Functional studies of thioltransferase (glutaredoxin) were also performed in several laboratories, mainly in Mannervik's group. Mannervik, 2; a1. (8) reported that the synthesized mixed disulfide between lysozyme and GSH (or Cysteine), or trypsin and GSH were reduced in the presence of thioltransferase, but not GSSG reductase and NADPH. This group further reported that thioltransferase could reactivate pyruvate kinase which had been inactivated previously by treatment with GSSG (132), and fully protect the enzyme from oxidative inactivation in air in the presence of GSH, GSSG reductase, and NADPH (133). The thioltransferase and thioredoxin systems were compared with regard to reduction of various disulfide substrates. Thioltransferase was more efficient in reducing small molecular weight disulfide, whereas thioredoxin had a higher affinity for protein substrates (134). A Japanese research team observed that thioltransferase could restore the activity of papain, which was previously inactivated by oxidation, in the presence of GSH, ig.liggg (135). Goswami 2E,§l, (24) described a cytosolic protein (glutaredoxin) having GSH-disulfide "transhydrogenase" "' ‘ act.-. micros ’1 I, reoc the es EEZVES assig: labor; disul reduc times thior (62), RRase viabl medlt RpasE SOUrC Calf prefie bone rabtd no: 17 activity that could activate microsomal iodothyronine deiodinase, a microsomal enzyme that catalyzes the in_yi£g9_conversion of thyroxine (T4) to triiodothyronine (T3) in the presence of GSH. Recently, it was reported that the thioltransferase and thioredoxin systems could protect the essential thiol groups of ornithine decarboxylase, the rate-limiting enzyme in polyamine biosynthesis (136). A novel function was recently assigned to mammalian thioltransferase as well as PDI in this laboratory, namely GSH dependent dehydroascorbate reductase activity (60). Glutaredoxin (thioltransferase) is a general GSH dependent thiol disulfide oxidoreductase capable of interacting with ribonucleotide reductase (RRase). The affinity of E; ggli glutaredoxin to RRase is ten times higher than that of thioredoxin, whereas the concentration of thioredoxin in these cells is ten times higher than that of glutaredoxin (62). However, neither thioredoxin nor glutaredoxin is essential for RRase activity because an E. £21; mutant, lacking both enzymes, was viable, but required supplemental cysteine for growth on a minimal medium, indicating the presence of a third unknown electron donor for RRase (62). Mammalian glutaredoxins (thioltransferases) from different sources display different behavior as electron donors for RRase. The calf thymus enzyme is active in the homologous RRase system in the presence of GSH, GSSG reductase, and NADPH (137), but neither the rabbit bone marrow enzyme nor pig liver enzyme served as electron donor for rabbit bone marrow RRase, and rabbit bone marrow thioredoxin also did not play such a role (123). These results raise the question, what is the electron donor for RR in rabbit bone marrow and pig liver? What roles do the two proteins play, i3 vivo? Generally, thioltransferase 1“ p... k-J beet dffi .4. are Est; Sit! 18 may play a role in the regulation of the biological activity of the thiol status of proteins or enzymes by reversible modification, but there is a shortage of meaningful ig_yi1g data. Now, it is generally speculated that the membrane associated PDI functions mainly on protein thiol-disulfide rearrangement, whereas both thioredoxin and thioltransferase regulate some preferred protein (enzyme) thiol status using different electron donor systems. For low molecular weight disulfides thioltransferase mediated reactions are the most likely (131). It is of interest that no thioltransferase (glutaredoxin) has been reported in plants so far, although a DHA reductase, quite different from thioltransferase in properties, has been described (138). The three established thiol-disulfide oxidoreductases contain an active site with the sequence of Cysxnys, i.e., the two cysteines are separated by two amino acids in a 14 atom loop. As stated above, there are two sequences, Cys-Gly-His-Cys, in PDI although it is not established whether one or both represent the active site or active sites, respectively. The active sites of thioredoxin and thioltransferase were identified as Cys-Gly-Pro-Cys (137) and Cys-Pro- Tyr(Phe for pig enzyme)-Cys, respectively. Two more cysteines, separated by three amino acids, are found near the C-terminus of thioredoxin and thioltransferase in mammalian tissues. The function of the second pair of cysteines is not clear. The active site and reaction mechanism of the two low molecular weight enzymes have been studied in recent years. Holmgren's group (139) identified Cys32 as the active site of E, ggli thioredoxin by kinetic studying of the alkylation reaction between the reduced protein and iodoacetic acid (1AA) or iodoacetamide (IAM) and by the radioactive labeling Cys” has of 8.3. mutant 1 to inve The mu: thiore. Val at enzyme CFS an l9 labeling of the active site. They found that the sulfhydryl group of Cys32 has a low pKfi_l_i_ JMlOl via transformation of the competent cells. The amplified recombinant M13mp18 RF DNA and single stranded DNA were prepared essentially as described (32). The RF DNA was digested with EcoRl (2 Units/ug DNA) again and analyzed on 5 2 polyacrylamide gel electrophoresis to confirm that the thioltransferase cDNA was inserted. In addition, the M13mp18- thioltransferase recombinant RF DNA was digested with Pstl and the largest enzymatically generated DNA fragment (M13mp18 + 3' region of the cDNA) was isolated by polyacrylamide gel electrophoresis. E. _c_o_l__i_ JMlOl was transformed with the religated M13mp18 containing the partial thioltransferase cDNA. The whole length cDNA and the 3' region of the 41 cDNA. were sequenced by Sanger's method (33) under the conditions ”S recommended. in BRL's KiloBase Sequencing Systen1 (34) using -dATP (Amersham) as the labeled nucleotide. 42 RESULTS AND DISCUSSION Antibody Screening of Thioltransferase cDNA-- A commercial (Clontech Laboratory Inc.) pig liver cDNA library in l-gtll was screened using rabbit polyclonal antibodies against pig liver thioltransferase and three positive clones were observed in 3.5 X 105 recombinants. Separate rescreening of the three clones with the same antibody in a low pfu density (about 200 plaques/plate) revealed that only two of them gave a number of positive signals. During the primary screening, we noticed that the color of the false positive signal was weaker than that of the other two. This phenomenon might be caused by the nonspecific binding of the antibodies and bacterial proteins, though the rabbit antiserum was pre- treated with E, coli extract to decrease the background. At time same time, a control experiment using pure A-gtll clones containing chicken ovalbumin DNA sequences (Clontech Laboratories Inc.) was performed. We treated these clones using antibody against chicken ovalbumin and the anti-thioltransferase antibody as the primary antibody separately, only the former showed positive signals, whereas the latter were negative. Accordingly, the antibody against pig liver thioltransferase (18) used in the present study has a reasonably high specific affinity and the two positive clones obtained by the antibody-screening appeared reliable. Oligonucleotide-Probe EHybridizations-- To confirm the positive clones screened by antibody, three Oligonucleotide probes (Table II) which were labeled with 32F at the 5'-end were used to hybridize to the two positive clones. Both of the clones gave positive hybridization reactions with the three probes (data not shown). Because each of the probes is an Oligonucleotide mixture which contains the nucleotide sequences complementarytx>all the possible codon combinations for its corresponding 43 amino acid sequence, the probes were expected to hybridize to the two clones if they contained the pig liver thioltransferase cDNA. In addition, the three peptide sequences selected for the probes were located near the N-terminus, at the middle and near the C-terminus of pig liver thioltransferase, respectively. After successfully hybridizing the three probes to the clones separately, this sequence distribution helped to establish that the cDNA inserted into the l-gtll clone was likely a complete rather than partial sequence. The hybridization experiment showed that the two antibody-positive clones were hybridized by all three Oligonucleotide probes, whereas the pure chicken ovalbumin cDNA was not hybridized by any of the probes. These results verified that two A-gtll clones having the full-length thioltransferase cDNA were obtained. Eggggrn-blotting Analysis the Fugion protein-- One of the positive clones was further analyzed by western blotting of the thioltransferase-B- galactosidase fusion protein produced by this clone. The pig liver thioltransferase cDNA was inserted into the A-gtll Eggz gene, coding for B-galactosidase, at the EcoRl site (Clontech Product Profile). When the recombinant A-gtll infects its host cell, the thioltransferase-B- galactosidase fusion protein, the product of the recombinant EggZ gene, will be made (35). The expression vector l-gtll makes a temperature- sensitive repressor (cI857) which is inactive at 42°C and has an amber mutation ($100) which only lyses the hosts containing the amber suppressor supF (25). On the other hand, E. gg_l_i_ Y1090 has a lac repressor which inhibits _l_a_c_Z gene expression until derepressed by IPTG, a deficient Egg protease which decreases the degradation of the recombinant fusion protein, and §_1_1p_F which suppresses the phage mutation ($100) (21). Thus, A-gtll will not lyse Y1090 at the incubation temperature of 30°C because 44 of its c1857 mutation, and EggZ won't be expressed until the addition of IPTG. ‘Taking advantage of these characteristics, we successfully induced the Y1090 lysogen strains by infecting the cells with the recombinant l- gtll and obtained sufficient thioltransferase-B-galactosidase fusion protein for western blotting analysis (Fig. 2). The fusion protein producedtnrthe recombinant A-gtll with the inserted thioltransferase cDNA will have a higher molecular weight than that of E,_ggEE B-galactosidase alone and react with both the antibody against pig liver thioltransferase and the antibody against E,ggEE B-galactosidase. Indeed, immunoblotting analysis of the fusion protein gave these results (Fig. 2). Bands in Panel A were visualized by Coomassie blue‘staining, while the bands in Panel B and C were visualized by treating with the anti-thioltransferase and the anti-B-galactosidase (Bio Rad), respectively. It can be seen that the fusion protein (Lane 2 in Panel A, B and C) has a higher molecular weight than E._cpEi_ B-galactosidase (Mr = 116.3 kDa) (Lane 1 in Panel A and C) and reacts with the anti-thioltransferase (Lane 2 in Panel B) and the anti-B-galactosidase (Lane 2 in Panel C). In contrast, the Y1090 control (Lane 3 in Panel A, B and C) had no bands corresponding to the fusion protein. Therefore, the western-blotting analysis of the fusion protein presents further strong evidence that we had obtained the full-length pig liver thioltransferase cDNA in A-gtll. Nucleotide Sequence AnalygEg-- The positive recombinant A-gtll clone containing; pig liver thioltransferase cDNA, as judged by antibody- screening, Oligonucleotide hybridization and western-blotting analysis, was amplified, and the recombinant A-gtll DNA was purified (see Experimental Procedures). The purified DNA was digested with EcoRl and the enzymatic DNA fragments were separated on a 5 Z polyacrylamide gel 45 Fig. 2 Western blotting analysis of the fusion protein. The preparation of the fusion protein and the performance of western blotting were described in the Experimental Procedures. Panels A, B and C wmre visualized with Coomassie Brilliant Blue staining, polyclonal antibodies against thioltransferase and antibody against B-galactosidase respectively. In Panels A, B and C: lane 1, Bio-Rad high molecular weight protein standard, the second band from the top also serves as a B- galactosidase positive control: Egne 2, 13. coli Y1090 lysogen extract (fusion protein); lane 3, E. coli Y1090 extract (as negative control): lane 4, pig liver thioltransferase. Lane 5 in Panel A, Bio-Rad low molecular' weight protein standard. FP, B-Gal and 'T.T. indicate the positions of fusion protein, B-galactosidase and thioltransferase, respectively. A ma I 2 3 4 5 l FP__2m“‘: ' a-.. 5% -- 427-.. “'5 -- - 3 H ‘0 21.5- -- 14.4— N-. D f 46 FP \p-Gau 47 Fig. 3 Polyacryggmide gel electrophoresis isolation of pig liver thioltransferase cDNA. The A-gtll/thioltransferase recombinant DNA was digested with EcoRl and the enzymatic fragments were separated in 5 2 polyacrylamide gel for 1 hour at a constant current of 30 mA. nggE, DNA molecular size marker (pBR322/HpaII), _L_a_g_e___2_, empty; Lane 3 and 4, 1- gtll/thioltransferase recombinant DNA (50 ug/lane). T.T.cDNA indicates the position of thioltransferase cDNA. 48 " _T.T. cDNA 49 (Fig 3). The length of the cDNA was expected to be less than 1000 base pairs since pig liver thioltransferase only has 105 amino acids (12) whereas l-gtll consists of 43.7 Kb and has only one EcoRl site (25). The lowest band (Lane 3 and 4 in Fig 3) had approximately 630 base pairs and was therefore a likely candidate for the thioltransferase cDNA. This fragment was subcloned into a M13mp18 at the EggRI site, and the insertion was confirmed by EggRI digestion of the recombinant M13mp18 DNA and analysis on a 5% polyacrylamide gel (data not shown). The corresponding single stranded M13mp18 DNAs, as the template, were sequenced by using the dideoxy chain termination method (33, 34), and the sequencing strategy was shown in Pig. 4, The whole nucleotide sequence of the cDNA for pig liver thioltransferase is shown in Fig. 5. The EcoRl recognition sequence, -GAATTC- (5'-3'), at both the 5' and 3' ends, was found, and the whole length of the cDNA was 635 base pairs (including the two EcoR] sites). The open reading frame has 321 base pairs starting with the initiation codon, ATG, and ending with the termination codon, TAA. The open reading region is flanked by two untranslated regions, an upstream sequence (23 base pairs) and a downstream sequence (291 base pairs). The downstream noncoding region has a long poly(A) tail of 57 A residues. Instead of the common poly(A) signal, AATAAA, we found the sequence, ATTAAA, 21 base pair upstream from the poly(A) tail. This indicates that a single base difference in the sequence, AATAAA, still provides the appropriate signal for poly(A) addition (36). The amino acid sequence of thioltransferase deduced from the open reading frame is the same as the previously reported amino acid sequence of pig liver thioltransferase (12) except at the N-terminus. The previously reported N-terminal amino acid sequence was Ac-Gln-Ala-Ala- (Fig. 6) which was 50 Fig. 4 Egg liver thioltrgnsfergse cDNAE§equencing strategy. The dark bar represents the coding region of the cDNA and the straight line represents the 5'- and 3'- noncoding regions. The vertical arrows indicate EcoRl or Pstl sites used in the cDNA subcloning and sequencing. The horizontal arrows indicate the direction and extent of sequencing. 51 EciRl P311 P511 5mm 3 ‘ a -r 4 V .L. 52 Fig. 5 Nucleotide sequence of pig livgr thioltransferase and EE§_deducedgggino ggidggequence. The nucleotides are numbered from 5' to 3' at the right of each row, and the amino acids are numbered in brackets from N to C termini. The poly(A) tail signal sequence, ATTAAA, is boxed. EcoRl sites are underlined: nucleotides in parentheses are not included in the 635 nucleotides of the cDNA. ATG Me t GTA Va 1 GAG Glu GTG Val C'I'G Leu GGT Gly AAG Lys AAA Lys GCT Ala G'I'l‘ Va 1 Leu CAT Asp CAA Gln AAA Lys AGA Arg TAA * CAA Gln 'I‘I‘C Phe CTC Leu ATT Ile GAG Gln GAG Glu GGG Gly GCA Ala ATC I le AGC Ser ACA Thr CTC Leu TGT Cys GAG Glu TTI‘ Phe AAC Lys CAA Gln GCC Ala ACA Thr ATA I 1e CTC Leu GTG Va 1 CCC Pro TTG Leu ACC Thr GCA Gly GGT Gly TTG Leu AAC Asn ACC Thr CCC Pro AGT Ser GCC Ala GGA Gly AGC Thr 53 AGC Ser TGC Cys TTC Phe GAC Asp ACA Ars TGC Cys CGC Arg WWCGCCTGTCAGC GGGAAGGTG AAA Lys ACT Thr CTG Leu ATC CAG CCT Ile TTC Phe GAA Glu AAC Asn GTA Va 1 CAT Asp CAG Gln Gln TGC Cys GGG Gly GAG Glu CCT Pro CTA Leu CAA Gln Pro AGA Arg CTT Leu A'I'l‘ Ile CGG Arg GAA Glu ATT Ile Gly AAG Lys CTG Leu CAA Gln GTC Val AGT Ser GGA Gly Lys ACA Thr GAA Glu CAT Asp T'l'l‘ Phe ATG Met GCT Ala Val CAG Gln TTT Phe TAT Tyr ATG Ile CAC Hi s CTG Leu TTA CAGCGAGGCAGACCCAAGCTGATAGCTCCCTGTAGAGCTGGATGGCA GTGCAGATAATGACAGCGC‘I'I‘CCTGGTGGATGGATGCCGGGCTACCTI‘CACTCAGCTGC AACTACTGTT'I‘ACTTAAAAATTCTGAAATGTGTTAACCCAAATAATTGGGGGGAGTGGG TTTTGGGGGACAAAACAGATTTTTCTTCTGACTCTGTI TGCCCC(A)SICGG( AATTC) [HE IAGTGGAATCAATCT 23 68 [15] 113 [30] 158 [45] 203 [60] 248 [75] 293 [90] 338 [105] 394 453 512 569 635 54 interpreted by conventional fast atom bombardment mass spectrometry (PAB- MS) examination of the T1 peptide, a tryptic octapeptide (12). This initial assignment was based on the observation of peaks at m/z 171 and at m/z 242 in the FAB-MS spectrum which were interpreted as representing Bl and B2 fragment ions, respectively. However, it was subsequently determined that the peak at m/z 171 represented extraneous material in the sample and did not represent a 81 ion from the octapeptide. In contrast, the N-tenminal amino acid sequence deduced from the cDNA sequence, which was clearly shown in the sequencing gel film (Fig. 7), was Met-Ala-Gln. The final N-terminal sequence of Ac-Ala-Gln appearing in pig liver thioltransferase results from the post-translational cleavage of the Met and modification of the Ala by acetylation. Glutaredoxin from calf thymus (17, 37) and rabbit bone marrow (38) has also been sequenced.and found to have the same N-terminus, N-acetylalanylglutamine. Thus, thioltransferase and glutaredoxin appear to be two names for the same protein based on similarity of amino acid sequence, immunochemical cross-reactivity, and other enzyme properties. Although the g1utaredoxin.gene (ggg) of E, ggEE_ has been cloned and characterized in Ml3mp9 (39), the present study represents the first successful cloning and sequencing of the cDNA for a mammalian thioltransferase (glutaredoxin), and now provides a powerful tool to further study the structural and functional relationships of the pig liver enzyme . 55 Fig. 6 Comparison of thg N-terminal amino acid seguences of Egg liggr thioltransferaseI calf thxmgs and rgbbit bone ggrrow glutaredoxin. The nonmatched amino acid (Glu) is boxed. T.T., thioltransferase; er, glutaredoxin. * Sequence obtained by amino acid sequencing (12); 6 Sequence deduced from the cDNA sequence (this study). Pig T.T.* Pig T.T.@ Calf er Rabbit er (Ac-)Gln (Met)A1a (Ac-)Ala (Ac-)Ala Ala Gln Gln Gln 56 Ala Ala Ala Glu Phe Phe Phe Phe Val Val Val Val Asn ASH Asn ASH Ser Ser Ser Ser Lys Lys Lys Lys 57 Fig. 7 Autorgdiogrgphy of the 5'-nucleotide sequence of the cDNA for pig liver thioltggnsfergse. From the bottom to the top is the direction of 5' to 3'. The 5'-EcoR1 recognition sequence and the codons for the N-terminal amino acids of thioltransferase are indicated on the right. 58 a n a A m N In. 13 ACGAACTCGGTA ACGT 10. 11. 12. 13. 14. 15. l6. 17. 18. 19. 59 REFERENCES Mannervik, B. and Axelsson, K. (1975) Biochem. J. Eg2, 785-788 Axelsson, K., Eriksson, S. and Mannervik, B. (1978) Biochemistry _ll, 2978-2984 Mannervik, B., Axelsson, K., Sundewall, A.-C. and Holmgren, A. (1983) Biochem. J. EEg, 519-523 Nagai, S. and Black, S. (1968) J. Biol. Chem. Egg, 1942-1947 Racker, E. (1955) J. Biol. Chem. EEZ, 867-874 Hatakeyama, M., Tanimoto, Y. and Mizoguchi, T. (1984) J. Biochem. 2g, 1811-1818 Gan, Z.-R. and Wells, W. W. (1987) Anal. Biochem. EgE, 265-273 Larson, K., Eriksson, V. and Mannervik, B. (1985) Method Enzymol. EEg, 520-524 Ziegler, D. M. (1985) Ann. Rev. Biochem. gg, 305-329 Axelsson, K. and Mannervik, B. (1983) FEBS LETTERS EgE, 114-118 Hatakeyama, M., Lee, C., Chon, C., Hayashi, M. and Mizoguchi, T. (1985) Biochem. Biophys. Res. Commun, EEZ, 458-463 Gan, Z.-R. and Wells, W. W. (1987) J. Biol. Chem. Efl, 6699-6703 Gan, Z.-R. and Wells, W. W. (1987) J. Biol. Chem. 262, 6704-6707 Gan, Z.-R., (1987) Ph.D. Thesis, Michigan State University Parker, A.J., and Kharasch, N. (1959) Chem. Rev. g2, 583-682 Luthman, M. and Holmgren, A. (1982) J. Biol. Chem. EgE, 6686-6690 Klintrot, L. M., Hobg, J. 0., Jbrnvall, H., Holmgren, A. and Luthman, M. (1984) Eur. J. Biochem. Egg, 417-423 Gan, Z.-R. and Wells, W. W. (1988) J. Biol. Chem. Egg, 9050-9054 Goswami, A. and Rosenberg, I. N. (1985) J. Biol. Chem. Egg, 6012 -6019 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 36. 37. 60 Yang, Y., and Wells, W. W., (1988) J. Cell Biol. E_Z, 747a Young, R. A. and Davis, R. W. (1983) Science, EEE, 778-782 Blake, M. S., Johnston, K. H., Russell-Jones, G. J. and Gotschlich, E. C. (1984) Anal. Biochem. Egg, 175-179 Matteucci, M.D. and Caruthers, M. H. (1981) J. Am, Chem” Soc., Egg, 3185-3191 Woods, D. (1984) Focus, Vol.6, No.3, Pl-3, Published by BRL Huynh, T. V., Young, R. A. and Davis, R, W. (1985) in DNA Cloning-a practical approach, Vol.1, pp 49-76, IRS Press, Oxford Maniatis, T., Fritsch, E. F. and Sambrook, J. (1982) in Molecular Cloning: A Laboratory Manual Laeulnli, U. K. (1970) Nature 227, 680-685 Towbin, H, Staehlin, T. and.Gordon, J. (1979) Proc. Natl. Acad. Sci. USA, 1g, 4350-4354 Ziai, M. R., Giordano, A., Armandola, E. and Ferrone, S. C. (1988) Anal. Biochem. EZE, 192-196 Ogden, R. C. and Adams, D. A. (1987) Methods in Enzym. EgE, 61-87 Wallace, D. M. (1987) Methods in Enzym. EgE, 41-48 Messing, J. (1983) Methods in Enzymn EgE, 20-78 Sanger, F., Nicklen, S. and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA, 1g, 5463-5467 Stambaugh, K. and Blakesley, R. (1988) Focus Vol. 10 No. 2, Published by BRL 35. Koenen, M., Griesser, H.-W. and Muller- Hill, B. (1985) in DNA Cloning-apractical approach (Glover, D. M., ed.) Vol.1, pp. 49-77, IRS Press, Washington, DC Lewin, B. (1987) in Genes III, p. 488, John Wiley & sons, Inc. Papayannopoulos, I. A., Can, Z.-R., Wells, W. W., and Biemann, K., 38. 39. 61 (1889) Biochem. Biophy. Res. Commun. _g2, 1448-1454 Hopper, S., Johnson, R.S., and Biemann, K. (1988) J. Cell Biol. E_Z, 4143 39. H663, J. 0., von Bahr-Lindstrom, H., J6rnvall, H., and Holmgren, A. (1986) Gene, gg, 13-21 62 CHAPTER III HIGH-LEVEL EXPRESSION OF PIG LIVER THIOLTRANSFERASE (GLUTAREDOXIN) IN ESCHERICHIA COLI 63 SUMMARY We report the first high-level expression of a mammalian thioltransferase (glutaredoxin) in Escherichia coli. A NggI site (CCATGG) was introduced into an cDNA encoding pig liver thioltransferase (glutaredoxin) by site-directed mutagenesis, in which the first G of the original sequence, GCATGG, was replaced by a C. The altered cDNA was cloned into an expression vector, plasmid pKK233-2, between the unique 5921 and EEngII sites and expressed in E. ggEE_JM105 at a high-level (8% of total soluble protein) after-6 h‘of isopropyl-B-D-thiOgalactopyranoside induction. The soluble and unfused product was measured by the thioltransferase thiol-disulfide exchange assay and immunoblotting analysis. The recombinant enzyme*was purified to a single band as judged by SDS-polyacrylamide gel electrophoresis and isoelectric focusing. The amino acid composition of the expressed enzyme agreed with that of the known sequence of pig liver thioltransferase (glutaredoxin). N-terminal sequence analysis revealed that unlike the native pig liver protein which is N-acetylated, the unfused recombinant enzyme was unblocked at the N- terminus (alanine). Various kinetic properties of the recombinant enzyme with regard to the exchange reaction were identical with those of the native enzyme. 64 INTRODUCTION Thioltransferase, also known as glutaredoxin, is a cytosolic enzyme that catalyzes the reduction of.tm 5:69.; 8:545 833.5 .9345 Fm _ _ _ _ _ _ _ _ 0 O O 0 O O 0 0 O O m 9 8 7 6 5 4 3 2 I own: 0 $552.: co 33:3. eszsem 114 DISCUSSIONS The primary structure of thioltransferase (glutaredoxin) has been reported for E. ggli (27), calf thymus (15, 16), pig liver (13, 14), rabbit bone marrow (11), and recently yeast (28). All the proteins have an active site of Cys-Pro-Tyr(Phe for pig enzyme)-Cys-, while the three mammalian enzymes contain an additional pair of cysteines near the C— terminus (Fig. 17). The sequences of the two regions containing the cysteine pairs are highly conserved in thioltransferases (glutaredoxins). The first region is the active center for each enzyme and the sequences in this region are identical except that a Phe instead of a Tyr was found in the pig enzyme, and only one basic amino acid is located in this region for the E. pp]; and yeast enzymes. In the second conserved region near the C-terminus, the sequences are the same in the three mammalian enzymes except that a Thr is replaced by a Ser in the rabbit enzyme. Despite the lack of the extra pair of cysteines, the E. Eli- and the yeast enzyme still have considerably high sequence homology to the mammalian proteins in this region suggesting that the second conserved region might have a structural function. However, our data showed that the replacement of the second pair of cysteines in the pig enzyme affected its activity only slightly. It is interesting that similar cysteine pair distributions occur in thioredoxin, another low molecular weight protein catalyzing various thiol-disulfide exchange reactions, i.e. there are two pair of cysteines in mammalian enzymes and only one pair in the active center of the bacterial and yeast thioredoxins (29,30). The present work provides the first direct evidence for the identification of the essential amino acids in the active center of 115 Fig. 17 Comparison of a_mino pcid sequences in two conserved regions. The amino acid sequences of thioltransferases from E. ppl_i_, yeast, calf thymus, rabbit bone marrow, and pig liver are compared in two cysteine containing regions. Alignment is based on the active site and the locations of the two regions are indicated with the number for each enzyme. The sequence between the two regions is omitted. Identical amino acids are outlined by solid lines, whereas dashed lines denote differences. 116 3:3 3:3 3:3 - eased _ e2 5. ”be be be new.“ so be be see. .llllL :5. ”be be be _P-- be be 2F as. be be on o: o: e: 0: sense £30 €96 n». 'ine "-" L I $3 $3 wp< m2 e? o: wh< 93 3» WNm m be he _eee , 8950 5C. 05 8&0 5 EA :30 SH. 95 95 96 who who £0 who be Beam use seen» seem 117 mammalian thioltransferases (glutaredoxins). Based on the proposed active site, we originally designed and synthesized six mutagenic oligonucleotides (Table I, oligo 1, 2,and 4-7) to create six mutants by site-directed mutagenesis. The surprising result from the ETT-Serzs mutant prompted us to create a seventh mutant, ETT-Ala”. The substitution of certain original amino acids caused large pl shifts, thus the pll of the buffers used in the purifications had to be modified (Table II). The pKa value of the sulfhydryl group of Cys22 (pK. = 3.8) is much lower than that of normal cysteine (p1(‘ = 8.5:0.5) (31), and Cys22 was speculated to be the active site of thioltransferase (19). This was 22 directly confirmed by results of changing Cys to Sern, which totally eliminated the enzyme activity. These data also showed that the amino 26 27 acids, Arg , and Lys are required for optimal enzyme activity since exchange at any of these positions generally decreased the activity with 25 with Serzs increased rather than the exception that replacement of Cys decreased the enzyme activity. This discovery necessitated a reevaluation of the enzyme mechanism for the mutant enzymes not capable of forming an intramolecular disulfide. Individual replacement of 2° or Lysn with Val26 or Gln” Arg , led to a relative 32% or 67% enzyme activity, respectively, but altering the two basic residues together with two neutral amino acids caused a cooperative loss in activity. How the two basic amino acids influence the catalytic activity was addressed in a companion paper (32). We did not try to change the two amino acids, Pro and Phe, between the two cysteines at the active center. However, two similar studies in T4 (33) and E. coli (34) thioredoxin were reported recently. Joelson, e_.t_ pl. (33) created three mutants, Cys—Gly-Pro-Cys, Cys-Val-Pro— 118 Cys, and Cys—Gly-Tyr—Cys, at the active site of T4 thioredoxin which has the sequence, Cys-Val—Tyr—Cys, and found no significant changes in the enzyme activity. Gleason _e_g Q. (34) constructed two mutants, Cys- Gly-Arg-Pro-Cys and Cys-Ala-Cys, at the native protein active site, Cys- Gly-Pro-Cys, of E. _c_gli_ thioredoxin by altering the size, and demonstrated that the longer mutant lost 85% of its activity, whereas the one with a shorter chain had no activity. The mutation studies implied that the distance rather than the specific amino acids substituted between the two active site cysteines is important, and the 14 atom disulfide loop at the active site of the oxidized enzyme seems to have been the preferred choice during evolution. However, as we demonstrate here, a serine at position 25 might have been expected to enjoy an evolutionary advantage. Acknowledgments--We thank Joseph Leykam, Director of the Macromolecular Structure Facility, Michigan State University, for synthesizing the oligonucleotides and Carol Smith for typing the manuscript. 1. 2. 3. 4. 6. 7. 9. 10. 11. 12. 13. 14. 15. 16. 17. 119 REFERENCES Racker, E. (1955) J. Biol. Chem. 2_11, 867—874 Holmgren, A. (1976) Proc. Natl. Acad. Sci. U.S.A. 13, 2275-2279 Nagai, S., and Black, S. (1968) J. Biol. Chem. LL34 1942-1947 Axelsson, K., Eriksson, S., and Mannervik, B. (1970) Biochemistry 11, 2978-2984 Hatakeyama, M., Tanimoto, Y., and Mizoguchi, T. (1984) J. Biochem. (Tokyo) §_5_, 1811-1818 Eriksson, S., and Mannervik, B. (1970) FEBS Lett. 1, 26-28 Larson, K., Eriksson, V., and Mannervik, B. (1985) Methods Enzymol. 13, 520-524 Gan, Z.-R., and Wells, W. W. (1987) Anal. Biochem. _1.§_2_, 256-273 Holmgren, A. (1979) J. Biol. Chem. 1544 3664-3671 Luthman, M., Holmgren, A. (1982) J. Biol. Chem. ES, 6686-6690 Hopper, S., Johnson, R. S., Vath, J. E., and Biemann, K. (1989) J. Biol. Chem. 2_6_4_, 20438-20447 Askeliif, P., Axelsson, K., Eriksson, S., and Mannervik, B. (1974) FEBS Lett. E8, 263-267 Gan, Z.-R., and Wells, W. W. (1987) J. Biol. Chem. 2_6_2, 6699-6707 Yang, Y., Gan, Z.-R., and Wells, W. W. (1989) Gene _8_3_, 339-346 Klintrot, L. M., Héog, J.-O., Jornvall, H., Holmgren, A., and Luthman (1984) Eur. J. Biochem. _1__4_4, 417-423 Papayannopulos, I. A., Gan, Z.-R., Wells, W. W., and Biemann, K. (1989) Biochem. Biophy. Res. Commun. 159, 1448-1454 Gan, Z.-R., and Wells, W. W. (1988) J. Biol. Chem. 263, 9050-9054 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 120 Mannervik, B., Carlberg, 1., and Larson, K. (1989) in Glutathione: Chemical, Biochemical and Medical Aspects (Dolphin, D., Poulsen, R., and Avramoviv, 0., eds.) Part A, pp 475-515, John Wiley 8: Sons, New York Gan, Z.-R., and Wells, W. W. (1987) J. Biol. Chem. 2E1, 6704-6707 Gan, A.-R, (1987) Doctoral Dissertation, Michigan State University. Yang, Y., and Wells, W. W. (1990) J. Biol. Chem. 2§_5_, 589-593 Zoller, M. J., and Smith, M. (1987) Methods Enzymol. _1_55_, 329-350 Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U.S.A. fl, 5463-5467 Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Second Edit. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York Carroll, S. B., and Laughon, A. (1987) DNA Cloning: a practical approach, Vol. 3, pp 89-111, IRL press, Oxford, Washington DC Laemmli, U. K. (1970) Nature 221, 680-685 H66g, J.-O., Jornvall, H., Holmgren, A. Carlquist, M., and Persson, M. (1983) Eur, J. Biochem. 1_3E, 223-232 Gan, Z.-R., Polokoff, M. A., Jacobs, J. W., and Sardana, M. K. (1990) Biochem. Biophy. Res. Commun. L6_8_, 944—951 Johnson, R. S., Mathews, W. R., Biemann, K., and Hopper, S. (1988) J. Biol. Chem. 2_6_3_, 9589-9579 Jones, S. W., and Luk, K.-C. (1988) J. Biol. Chem. E6_3, 9607-9611 Lindley, H. (1960) Biochem. J. E, 577-584. Yang, Y., and Wells, W.W. (1991) J. Biol. Chem., submitted for publication (Chapter V). 33. 34. 121 Joelson, T., Sjéiberg, B.-M., and Eklund, H. (1990) J. Biol. Chem. 265, 3183—3188 Gleason, F. K., Lim, C.-J., Gerami-Nejad, M., and Fuchs, J. A. (1990) Biochemistry 29, 3701-3707 122 CHAPTER V CHARACTERIZATION OF MUTANT PIG LIVER THIOLTRANSFERAS E 123 SUMMARY Seven mutant pig liver thioltransferases (glutaredoxins) were created by site-directed mutagenesis (Yang and Wells (1991) J. Biol. Chem. Submitted for publication). The properties of these mutants with respect to structure and function were analyzed and compared with those of the wild-type enzyme. The pI of each protein was measured in its reduced and oxidized form by isoelectric focusing and the resulting pl shifts of the mutants relative to the wild-type were compared. The optimum pH for the enzyme activity of the mutants was determined to lie between pH 8.5-9.0, similar to that the wild-type enzyme. The kinetic behavior of the enzyme activity with respect to substrate, S- sulfocysteine, concentration with a constant amount of GSH was analyzed. In order to test the influence of the amino acid exchanges on the pKa of the sulfhydryl groups of Cys22 , the active site of thioltransferase, the pK‘ values for the mutant enzymes were obtained by studying the second order kinetics of the alkylation reaction of equal molar concentrations of iodoacetamide and thioltransferase. Conclusive evidence was obtained that Cys22 is the active site of the enzyme, and the extremely low pK‘ value of its sulfhydryl group is facilitated 2i primarily by Arg . In addition, the second pair of cysteines, Sern and Seru, near the C-terminus, are not directly involved in the active center but they may play a role in defining the native protein structure. It is possible that C ys” is not necessary for enzyme activity, but an amino acid with the size and polarity, e.g., serine, is essential at this position. The role of Lys21 at the active center is different from that of Arg”, and may be important in stabilizing the ES intermediate by electrostatically attracting negatively charged groups of substrates (e.g. —COO' or -SO3' 124 -coo‘ or -so,‘ of Cys-so,‘ or the -coo‘s of GSH) with its positively charged side chain (~NH3+). Dehydroascorbate reductase activity of thioltransferase (glutaredoxin), (Wells et al. (1990) J. Biol. Chem. 2_65, 15361-15364) was measured for each mutant and compared with that of the wild-type enzyme revealing a 90% increased activity for the mutant, ETT-Serzs. These results are consistent with the suggestion that the 22 formation of an intramolecular disulfide bond between Cys and Cys25 is not required for enzyme activity of selected mutants. 125 INTRODUCTION Thioltransferase (Glutaredoxin) is a low molecular weight heat stable protein with activity catalyzing thiol-disulfide exchange reactions in the presence of GSH. These reactions are considered important in maintaining cellular thiol/disulfide equilibria (1-3). There are four half-cystine residues in all sequenced mammalian thioltransferases, and two of them are located in a catalytic center near the N-terminus (4-8), whereas there are only two cysteine residues in yeast (9) and E. p.911 (10). The active site of thioredoxin from E. 9_Q_I_i_ was identified to be Cys32 and to have a sulfhydryl pK. value of 6.7, with high sensitivity to alkylating reagents (11). Like thioredoxin, when incubated with iodoacetic acid or iodoacetamide, thioltransferase was inhibited by alkylation at its active site, Cys22 (12). The pK' of the Cys22 sulfhydryl group was estimated to be 3.8, much lower than a normal pK‘ of cysteine (8.510.5)(13). The extremely acidic pK' of the Cys22 thiol group is believed to be facilitated by its unique surrounding microenvironment, but which amino acid residues are involved in such actions is not established. We reported the high-level expression of pig liver thioltransferase in E. 9911, and showed that the recombinant enzyme was identical to the native one in most biochemical properties except the pI difference caused by the nonacetylated N-terminal alanine of the expressed protein (14). Recently, it was reported from this laboratory that thioltransferase, as well as protein disulfide isomerase, had intrinsic dehydroascorbate (DHA)1 reductase activity and may be involved in the major mechanism for the regeneration of ascorbic acid from dehydroascorbic acid in animal cells (15). In Chapter IV (16), we 126 described the creation of seven mutant pig liver thioltransferases with amino acid substitutions in the active center by site-directed mutagenesis and identified the essential amino acids for thiol-disulfide exchange activity. Cys22 was directly identified as the essential catalytic site since a replacement with serine at this position totally eliminated 28 the activity of the mutant protein; and Arg and Lys” were required to maintain full enzyme activity. An increase in activity by the 25 25 with Ser led to the suggestion that the formation 22 25 replacement of Cys of an intramolecular disulfide bond between Cys and Cys was not necessary for the catalytic mechanism (16). In this report, we evaluate the specific roles of the amino acids in the active center in the reaction mechanism by further characterization of the mutants including analysis of the pl values, kinetic behaviors, pH optima, pK‘ of the Cys22 sulfhydryl group, and the DHA reductase activities. 127 EXPERIMENTAL PROCEDURES Materials-- Isoelectric focusing gel and p1 marker proteins were from Serva; dithiolthreitol (DTT), NADPH, glutathione reductase, and iodoacetamide were from Sigma; 2-hydroxyethyl disulfide (HED) was purchased from Aldrich Chemical Co. Inc.; S-sulfocysteine (Cys-803') was prepared as described previously (17); Sephadex G-25 was from Pharmacia; dehydroascorbic acid was obtained from Fluka Chemika- Biochemika. All other standard reagents are analytical grade. Preparation of Reduced thioltpansferases" The wild-type and mutant pig liver thioltransferases were expressed and purified to homogeneity as described previously (14, 16). Each of the purified enzymes was incubated with 25 mM DTT in the presence of 100 mM sodium phosphate buffer, pH 7.5 at room temperature for 30 min. The excess DTT and the salts were removed by passing the incubation mixture through a Sephadex G-25 gel filtration column (1.5 x 70 cm) equilibrated with 20 mM sodium phosphate buffer, pH 7.5. The reduced protein was eluted with the same buffer used to equilibrate the column and was concentrated in a centriprep 10 concentrator (Amicon) by centrifugation. Each of the concentrated reduced thioltransferases was then divided into aliquots of 0.1 ml and stored at -70°C until used. Isoelectric Focusing Analysis of The Mutpnt Thioltransferases-—The reduced form and oxidized form of pig liver thioltransferases were obtained by incubating 1 ug of each purified enzyme with 10 mM DTT and 10 mM HED, respectively, at room temperature for 30 min. The incubation mixtures (8 111 each) were then directly loaded onto a pre- cooled Servalyt Precote isoelectric focusing gel (125 x 125 mm), pH 3-10. The electrophoresis of the gel was performed according to the 128 manufacturer’s instruction. Briefly, the gel was first run at a constant current of 5 mA until a power of 4 W was reached, then at a constant power of 4 W until 1700 volts were reached, and finally, the current remained at 1-2 mA for 10 min. The gel was fixed with a solution of 36% methanol, 6% trichloroacetic acid, and 3.6% sulfosalicylic acid, and was stained with Coomassie Brilliant Blue R-250. Optimum pH of the MM Thioltransferases-- The thiol-disulfide exchange activity of the wild-type and the mutant pig liver thioltransferases was measured over the pH range of 5.5-9.5 in a 500 pl mixture containing 0.5 mM GSH, 1.4 unit glutathione reductase, 2.5 mM Cys-803', 0.35 mM NADPH, 1.5 mM EDTA, 137 mM sodium phosphate buffer (pH 5.5-7.5) or 137 mM Tris-HCl buffer (pH 8.0-9.5). The amount of protein used in these experiments was different for each individual enzyme, depending on the relative catalytic activity detected. Eipetic Eehavio:-- Thioltransferase activity dependence on the concentration of Cys-803' was determined by the standard enzyme assay system with various amount of the substrate, in 137 mM sodium phosphate buffer, pH 7.5, and 1.5 mM EDTA, 0.5 mM GSH, 0.35 mM NADPH and 1.4 units of glutathione reductase. T e V es of Mut t and Wild-t e Thioltransferases-- Equal concentrations (60 11M) of the reduced thioltransferase, obtained as described above, and iodoacetamide were incubated in 100 mM sodium citrate buffer (pH 2.5-5.5) or 100 mM sodium phosphate buffer (pH 6.0- 9.0) at room temperature. At different time points, various amounts of incubating mixture were withdrawn, and the enzyme activities were assayed after appropriate dilution, i.e., the quantity of each enzyme used for activity measurement was different. The alkylation reaction of 129 equal concentrations of thioltransferase and iodoacetamide followed second order reaction kinetics, and the apparent rate constant, klPP’ of these reactions at each pH for each enzyme was determined. A plot of the dependence of kapp on pH gave an estimated pK. value for Cyszz of each mutant retaining a cysteine residue at the 22 position. The DHA Reductase Activity of Thioltransferase--The DHA reductase activity of the wild-type and each mutant thioltransferase was measured in a 500 pl mixture of 2 mM GSH, 1.5 mM DHA, 160 mM sodium phosphate buffer, pH 6.8, 1.2 mM EDTA, and 0.4 ug thioltransferase, spectrophotometrically, by monitoring the increase in absorbance at 265.5 nm and 30°C. One unit of DHA reductase activity is defined as that amount of enzyme catalyzing the formation of 1 nmol of ascorbic acid per min under the standard conditions. A blank control without enzyme was measured simultaneously with each group of activity assays. 130 RESULTS AND DISCUSSION Isoelectric Focusing Apalysis--The wild-type thioltransferase (ETT) 22 25 25 and mutant thioltransferases (ETT-Ser , ETT-Val“, 2'! 21 22 , ETT-'88P ’ ETT‘Ala ETT-Gln , ETT-ValuGln , and ETT-Ser -Ser°2) were analyzed both in their reduced forms (Fig. 18A) and oxidized forms (Fig. 188) by isoelectric focusing. In panel A, lanes 1 to 9 were loaded with Serva pl marker 22 25 21 proteins, ETT, ETT-Ser , ETT-Val”, ETT-Gln , ETT-ValuGlnn, 25 , ETT-Ser ETT-Ala , and ETT-Sern-Seru, respectively. In panel B, the pI of the oxidized forms of each protein are shown. Lanes 1 to 7 and lane 9 were loaded with the same enzyme as those in Panel A and lane 10 was ETT- Ala”. As seen in Fig. 18, the pl values of the reduced thioltransferases are 7.0 for ETT, ETT-Serfi, ETT-Ala”, and ETT-Sern-Sern, 7.5 for ETT- Seru, 5.8 for ETT-Val”, 6.1 for ETT—Gln", and 5.3 for ETT-ValuGlnn, whereas the pl values of the oxidized forms are approximately 8.0 for 25 25 "-Ser”, 8.4 for ETT-Sern, 7.0 for ETT, ETT-Ser , ETT-Ala , and ETT-Ser ETT-Val2° and Gln”, and 6.0 for ETT-Valfi-Glnn. These results clearly indicated that exchange of some amino acids, especially the charged residues (e.g., Arg and Lys), at the active sites caused the pI shifts of the proteins. Normally, when a protein is in its native folded form, the pI value of the protein is the sum of the net charges on the surface of the molecule (18). In the present case, all thioltransferases, including the wild-type and mutants, analyzed on isoelectric focusing gel were in their native folded forms. Thus, the pI values of these proteins obtained by this technique should reflect the total surface charges of these native molecules and the pI shift should follow the changes of the surface charged groups. The substitution of Cys with a Ser at 131 Fig. 18. Isoelectric focusing of mm. thioltransferggep. The purified wild-type and mutant thioltransferases (1 ug) were treated with 10 mM DTT (Panel A) or 10 mM HED (Panel B) for 30 min at room temperature, and the pI values were measured on a Servalyt Precotes IEF gel, pH 3-10, according to the manufacturer's instructions. The gel was stained with Coomassie Brilliant Blue. The reduced forms in Panel A, lanes 1 to 9, were Serva pI maker proteins, ETT, ETT-Sern, ETT-Ser's, 21 25 21 25 12 I2 ETT-Val“, ETT-Gln , ETT-Val Gln , ETT-Ala , and ETT-Ser -Ser , respectively. The oxidized forms in Panel B, lanes 1 to 10, were pI standards, ETT, ETT—Sern, ETT—Ser”, ETT-Val", ETT-Gln”, ETT-ValuGlnz', ll 22 ETT-Ala” ((0.1 ug), ETT-Ser -Ser , and ETT-Ala”, respectively. 132 In K00..O- 'tW@..O_ 1. 1. 1. Ram.” "hem I. .... “a: Home 1.. ill. 1. I onus. l. tomb .. cm a 1. l .5 1-03. t 1.1.03 m. I can ( t. . . .. no mean as 1 etc 1 “Lynn .1 00d “18¢ a 6302.033”... H teem mm senile. He 133 position 22 caused a pl change in both reduced (0.5 pH units) and oxidized (0.4 pH units) forms, however, the same substitutions at positions 25, 78 and 82 had no influence on the pl values (Fig. 18). These results provide additional evidence that Cys22 is the only cysteine residue in its thiolated form (~83 at a physiological pH, and that it is exposed or partially exposed to the molecular surface, whereas Cys”, '° and Cys'z are either in their sulfhydryl forms (-SH) or buried in Cys the protein core. The three dimensional structures of E. gel; (19) and T4 (20) thioredoxins have shown that their active sites are parts of a protrusions of the molecules. So it is possible that the active site of thioltransferase (glutaredoxin) is on the surface of the molecule, and only this location of the active center can explain how the low molecular weight enzyme interacts with its protein substrates (usually much larger than the enzyme in size), such as pyruvate kinase (1) and ribonucleotide reductase (21). Certainly, this proposal needs further support from the three dimensional structure of thioltransferase, and such studies are now in progress. There are two bands for oxidized ’5 and ETT-Ala” (Panel B of Fig. 18). We think the forms of ETT-Ser upper one is the unoxidized (reduced) form and the lower one is the oxidized form for both mutants. Striking pl shifts were observed in both reduced forms and oxidized forms when the two basic amino acid residues were replaced by neutral amino acids either individually or together. Compared with the wild-type enzyme (ETT), mutants of ETT-Val“ (Arg to Val), ETT-Gln” (Lys to Gln) and ETT-ValuGlnz' (Arg-Lys to Val-Gln) have more acidic p13, and the double mutant has the most acidic pI. These pl changes apparently resulted from the loss of the positively charged Arg and Lys 134 or both in the active center possibly located on the molecular surface. We showed in a previous paper that the enzyme activity of mutants of 21 21 ETT-Val", ETT-Gln and ETT-Val"Gln are only 32%, 67% and 5% of that 2' and Lys”, of the wild-type (16). Thus the two basic amino acids, Arg are very likely involved in the catalytic activity (see below). Optimum pH End Kinetic Behavior-- Thiol-disulfide exchange activities of the wild-type and the mutant enzymes were assayed over the pH range of 5.5-9.5 using different amounts of protein. The general pattern of thioltransferase activity dependence on pH for each enzyme was similar with low rates at acidic pHs and with maximum rates at pH 8.5-9.0 (Fig. 19). Thus, modifications at the active center did not change the optimum pH of the enzyme, excluding the totally inactive mutant ETT-Seru. These results imply that the amino acid substitutions at the active center did not cause major conformational differences and the activity changes were the result of the active site alterations. Future three dimensional X-ray crystallographic analysis should establish the validity of this speculation. The kinetic property of each thioltransferase with respect to the Cys-SO}. concentration is shown in Fig. 20. The v vs [S] plots of the wild-type and mutant thioltransferases showed non-Michaelis-Menten kinetics, i.e. at high substrate concentration, the enzyme activity was inhibited. The values of K05 for these enzymes were estimated to be 0.5-0.8 mM. Our unpublished data clearly showed substrate (Cys-803' or cystine) inhibition of the thioltransferases, and the products of thioltransferase action, cysteine and 11803., were inhibitors of glutathione reductase. But the latter inhibition could be neglected in the current 135 Fig. 19. Optimum pH of mutant Thioltransferases. TT activity dependence on pH was compared among the mutants and wild-type proteins. The activities of wild—type and mutant thioltransferases were measured by the standard assay system in which 137 mM sodium phosphate (from pH 5.5 to 7.5) and 137 mM Tris-Hcl (from pH 8.0 to 9.5) were used. In each assay, 0.25 ug ETT, 0.20 ug ETT-Ser's, 1.0 ug ETT- Ala”, 0.90 ng ETT-Val”, 0.4 ug ETT-Gln", 3.0 ug ETT-Valz'Glnz', and 0.27 18 ug ETT-Ser -Ser82 was used, separately. Each value is the average of three separate experiments. pMoIe NADP'Ymin 0.08 0.07 0.06 0.05 p i 0.03 0.02 0.01 0.00 , 136 EEZEIII ETT ETT-Ser25 ETT-A1625 e'rT-Vol26 ETT-Gina? ETT— V01266|027 ETT-Ser788er32 137 Fig. 20. Kinetic behgvior of mutant Thioltransferases. Thioltransferase activity dependence on S-sulfocysteine concentration was compared for the wild-type and mutant enzymes. The standard assay (see Experimental Procedures) was used with increasing 8- sulfocysteine concentrations. In each separate assay, 0.35 ug ETT, 0.35 pg ETT-Serz', 3.0 pg ETT-Ala", 0.85 pg ETT-Val“, 0.65. pg ETT-Gln", 4.5 21 12 22 pg ETT-Val"Gln , and 0.36 ug ETT-Ser -Ser were used, Each value is the average of three different experiments. 138 :25 o53m>o83m . m ON m._ _ O._ 0.0 «mammoscomrkhw 55083?er hue—OIFFm e~_e>1._L.w 32$»: exams: ._:_.m _0.0 NO. 0 Una/mow alowfi no.0 139 study because of the excess of glutathione reductase and the negligible concentration of cysteine and H803. at the initial stages of the reactions. The pKa Va_l:ues of the Exp_re3§ed Thioltransferase?- Thioltransferase activity can be irreversibly inhibited by iodoacetic acid '2, and the oxidized and iodoacetamide by alkylation of its active site-Cys (intramolecular disulfide or mixed disulfide) form of the enzyme is protected against the alkylating reagents (12). It is known that the alkylation of thiols only occurs on their thiolate (-S') forms, and this reaction is, therefore, a strongly pH dependent process (13, 22). Thus the enzyme activity measured during an alkylation reaction must be a measure of unalkylated reduced (free) thioltransferase. If the same initial concentration of reduced thioltransferase and iodoacetamide are incubated together, this carboxamidomethylation reaction should obey the kinetics of a second order reaction as shown: kt = [TT-AL] / [TT-SH] ([TT-SH] - [TT-AL]) or kt = 1 / ([TT-SH] - [TT-ALl) - 1 /[TT—SH] where k is the apparent rate constant, t is the time, [TT-SH] represents the initial concentration of reduced thioltransferase, and [TT-AL] represents the concentration of the alkylated enzyme. As indicated above, the item [TT-SH] - [TT-AL] reflects the amount of reduced enzyme which is the function of the thioltransferase activity measured at time t. The reduced wild-type and mutant thioltransferases (60 uM) were incubated with the same concentration of iodoacetamide (60 11M), separately, in the presence of 100 mM sodium citrate buffer (pH 2.5-5.5) or sodium phosphate buffer (pH 6.0-9.0) at room temperature. At the incubation times of 0, 5, 15, 25, 35, and 45 min, samples were withdrawn 140 and the enzyme activities were measured. At each specific pH, a plot of 1/([TT-SH] - [TT-AL]) (i.e., 1/thioltransferase activity) against time, t, gave a straight line and the slope of the straight line gave an apparent rate constant, klPP' of the alkylation reaction at a specific pH. One ’5 and 60 example is shown in Fig. 21, in which 60 11M of mutant ETT-Ser pH of iodoacetamide were incubated in 100 mM sodium phosphate buffer, pH 6.8 at room temperature, the plot of 1/enzyme activity vs t gave a straight line, the apparent rate constant, km, was 5.5 mM"min'1 and the corresponding half-time, tin, of the reaction was 3.0 min. Similar plots at various designated pHs for each enzyme were drawn (data not shown) and their k.” values were calculated. The pK‘ values of the Cys22 sulfhydryl group for each enzyme was obtained from the midpoint of plots of k.pp vs pH (Fig. 22). For the expressed wild-type thioltransferase (ETT), the apparent rate constant was pH dependent over the pH region of 3.0 to 4.5, whereas it was pH independent between pH 4.5-8.5. Since alkylation of thiols only occurs in the thiolate form (-S') (13,22), the extremely low klPP values below pH 3 indicated that 22 Cys was in the sulfhydryl form (-SH) and not sensitive to the alyklating reagent. The increasing k values between pH 3.0-4.5 ‘99 '2 sulfhydryl occurred in this pH signified that the deprotonation of Cys range and the thiolate forms reacted with iodoacetamide. The unchanged kill? values over the pH region of 4.5 to 8.5 implied that the maximum rate of alkylation reaction was reached and all Cys22 side chains were in the thiolate form. The pKa value of Cys22 of the wild-type recombinant thioltransferase was about 3.8, consistent with that of the native enzyme (12). Thus, the acetylation at the N-terminus of the native pig liver enzyme has no influence on the pKa of Cysz'. For the mutant enzymes, 141 Fig. 21. Ajlot of the alkylation reaction between the same 25 concentrations (60 11M) of reduced ETT-Ser gnd iodoacetamide. This reaction was performed at room temperature in 100 mM sodium phosphate buffer, pH 6.8. The values of reduced thioltransferase at various times were determined by enzyme activity assay after dilution (see text for , of the reaction details). The second order apparent rate constant, kapp was 5.5 mM"min'1 and the half—time was 3.0 min. 142 O p 240. 200 _ '7" 160 .. 2 E - "'3' 120 .. - t: 1— .. "" kapp‘ 5.5 mM"min" 80 .. V t 2= 3.0 min 40 _ l l L l I l l l l 5 10 I5 20 25 30 354045 TIME (min) 143 Fig. 22. pH dependence ofgspcond order apparent rate constants of the reactions between each of the reduced thioltransfegaseg (60 pM) pnd iodoacetamide (60 11M). The k‘pp values of the reactions were obtained as described in Fig. 4 and Experimental Procedures. kappth-nin“) 144 -—N01«b I —Nuhot -ueumg ETT-Set25 l l l I l ETT-Gln” j l l l l —NUA —N02-2> 0103 ETT-Stern Ser82 l I l J 2 23456789 pH 145 25 21 25 18 82 ETT-Ser , ETT-Gln , ETT-Ala , and ETT-Ser -Ser , the pH sensitive kapp values were in the regions of 4.0 to 6.0, 3.5 to 5.5, 4.5 to 6.5, and 4.0 to 22 5.5, respectively. The pK' values of the Cys side chain were estimated 21 to be 4.9 for ETT-star”, 4.3 for ETT-Gln , 5.9 for ETT-Ala”, and 4.4 for 18 ETT-Ser -Ser'2, all more basic than that of the wild-type. Substitution of the cysteine with either a serine or an alanine at position 25 caused a more basic shift of the pK. of Cys22 than the changes at position 27 21 18 82 18 82) 21 to Gln") and position 78 and 82 (Cys 28 (Lys -Cys to Ser -Ser Lys was speculated to facilitate the low pKa at Cys'z, but a 21 as well as Arg 21 change of Lys to Gln only slightly increased the pl(a of Cys22 (ca. 0.5 pH units) and decreased the activity by 33 % (16), whereas a greater 25 pKa increase was caused by the change of Cys to Serz', yet enzyme activity was raised 10% (16). Thus, for mutant ETT—Gln", the 33% loss of enzyme activity was not the result of the slight pK‘ increase, but Lys" likely played some other role in the enzyme catalytic mechanism. Currently, we do not know the function of Lysz', although one possibility is that this residue can stabilize the enzyme-substrate intermediate by ionic interactions between its positively charged side chain and a negatively charged group of the substrates, e.g., GSH. It is interesting '5 with Ser'5 and with Ala's, separately, resulted in 22 that replacing Cys different pKa changes (1.1 and 2.1 pH units, respectively) at Cys and resulted in totally different activity alterations (10% increase vs 91% decrease, respectively) (16). Compared with serine, the relatively more hydrophobic alanine replacing Cys at this position might disturb the 2' to 18 local three dimensional structure of the active center and cause Cys be less exposed. The exchanges of the two downstream cysteines, Cys 82 22 and Cys , with two serines had little influence on the pKa value of Cys . 146 12 group is not facilitated by either Cys”, Lys", or Cys and Cys'z. In contrast, the amino acid responsible for the low pK' at Cys22 was Arg". We could not measure the pK‘ value of mutants ETT-Val“ and ETT-ValuGlnt', because the apparent rate constants, kW of both mutants l were very low (about 0.4 mM'min") and pH independent over the region of 2.5 to 8.5., that is, the two mutants were not sensitive to iodoacetamide. These results clearly indicated that replacing Arg with Val2° significantly decreased the deprotonation of the active site sulfhydryl group. We conclude that the role of Arg28 is to facilitate the low pK' of Cysn, i.e., enhance its 8' nucleophilicity, necessary for the thioltransferase catalytic reaction. The DEA deggtgse Aggvity" In the presence of GSH, thioltransferase can catalyze the reduction of DHA to ascorbic acid (15). The intrinsic DHA reductase activity of the wild-type and each of the mutant enzymes was measured as described in Experimental Procedures and compared with each other based on the same amount of protein (0.4 ug) (Fig. 23). With the activity of the wild-type enzyme defined as 100%, the relative activities of the mutants were 0% for ETT-Sern, ETT- Vela-Gln", and ETT-Ala”, 194% for ETT-Ser's, 30% for ETT-Val“, 73% for 21 12 22 ETT-Gln , and 71% for ETT-Ser -Ser . Like the thiol—disulfide exchange 22 activity, the Ser mutant had no detectable DHA reductase activity. 22 This result indicates that Cys is very likely the catalytic site for both intrinsic activities. Compared with the thiol-disulfide exchange activity, 25 ETT-Ser had a significantly greater DHA reductase activity, whereas there was no detectable activity for ETT-Ala”. The evidence suggests that a serine is more favored than a cysteine at position 25, especially for DHA reductase activity. The DHA reductase activity of 147 Fig. 23. Comparison of the DHA reductpse activity of thioltransferases-- The DHA reductase activities of the wild-type and mutant thioltransferases were measured based on the same amount of proteins (0.4 ug) as described in Experimental Procedures. The activity of the wild-type enzyme was defined as 100%, and the relative activities of mutant enzymes were compared. N88028:!) o .— o 0 mm?o>'.khw 010 NNEOIFFM E mwmw.—y\'.nrirmw hurl-Au ONO-(IFS I nmeomtkhm Ill PPM I O O 10 O to CO NKO OOO OO‘tt'D 58-3 see :8 ‘1'8585. .—-——-' and CF? in 01 0.12 I! [g 21 Imo O N O V 9301919101110qu 10 Aignglov 91010193 165 reducing previously air—oxidized forms of the enzymes. When pretreated with the disulfide-like substrate, Cys-803., all enzymes were totally or partially protected from inactivation by IAM except the two Cyszs substituted mutants, ETT-Serzs and ETT-Ala” (Fig. 25, middle). Identical results were obtained using E-cystine instead of Cys-803 (data not 28 21 18 shown). ETT-Valz', ETT-Val Gln and ETT—Ser -Ser82 totally retained 1 their activity by pretreatment with Cys-803., possibly by the formation of an intramolecular disulfide (Reaction 6 and 7) which prevented IAM reaction with Cysn. ETT-Serfi and ETT-Ma's, lacking the ability to _E1— _2.—-_I:—-‘.‘..L"\v- .. . . 1 I. A. establish an intramolecular disulfide bond within their active center, were inhibited by IAM even after the initial formation of a mixed disulfide bond between the enzyme and the substrate (Reaction 10), and in the presence of an excess of Cys—SOs. (Cys-803‘ : IAM : enzyme = 2.5 mM : 0.12 mM : 0.06 mM). ,5 ,ssn E + asses—:1; + RSH (10) ‘su(on) SH(0H) ,S-SR ,s-sc E\ + GSH:E\ + RSH . (11) smou) smon) ,s-sc ,s’ E + 0311:13 + 0350 (12) ‘smom ‘sn(on) where (OH) represents the hydroxyl group of Serzs of mutant ETT-Serzs. This phenomenon can be explained on the basis that Reaction 10 is reversible. IAM could remove the enzyme from the reaction by reacting with free enzyme and forming a dead end complex, i.e., pull the reaction 21 to the left. For ETT and ETT—Gln , the mixed disulfide formed in Reaction 6 was competitively replaced either by the -SH group of Cys25 166 to form an intramolecular disulfide (Reaction 7) or by the products to reverse the direction of Reaction 6. In this situation, the rate of the reversal of Reaction 6 was much slower, i.e., less free enzyme was available to IAM, and these two enzymes were only partially inactivated by IAM. It is of interest that when simultaneously preincubated with Cys-803. and GSH, all thioltransferases were relatively well protected, including the two mutants lacking Cyszs (Fig. 25, right). We believe that the coexistence of both substrates made the enzyme accessible to GSH to 2' by replacing Cys25 (Reaction form an enzyme-8G mixed disulfide at Cys 8 or 11) or the mixed disulfide between enzyme and the initial disulfide substrate (Reaction 6 or 10). This newly formed disulfide bond may be relatively more stable than the enzyme-half-substrate disulfide bond and the final step of the process, Reaction 9 or 13, might be the rate determining step. It is likely that during the preincubation, the enzyme catalyzed reaction had reached a steady-state balance in which the enzyme was virtually saturated by the substrates, forming predominantly mixed and intramolecular disulfides, so that when IAM was added, little free enzyme was available. However, IAM gradually pulled some of the enzyme molecules from the system at a slow rate since partial inhibition of the activity was observed. The enzymes were also pretreated with HED, and, except for the two Cys25 substituted mutants, all others were fully protected against inactivation by IAM (Fig. 26). Because of the two exceptions, this protection appears to be acquired by the formation of intramolecular disulfides. Similar studies were done using the native pig liver thioltransferase, this enzyme was fully protected when preincubated with cystine, Cys-803', or HED, but not with GSH (data not shown) in «met-mags mm 167 Fig. 26 Pig liver thioltrpnsfergses protection by HED. The protection experiments were performed by pretreating each of the wild- type and mutant thioltransferases (0.06 mM) with 2.5 mM HED before the addition of 0.12 mM IAM. The test conditions were the same as described in the legend of Fig. 25. Each value is the average of two separate experiments. Relative Activity of Thioltransferase 100 90- 80 7o 60 so 40 30 20 10 168 o—O ETT I-I ETT- Ser25 H ETT-A1025 co ETT-V0126 A—o ETT-611127 0-0 ETT-V012661n27 o—o ETT- SerZBSerez l l l l 5 10 15 20 25 30 35 40 45 Time (min) 169 agreement with Gan and Wells (15). The only difference between the native and the recombinant enzyme is that the former has a N-acetylated N-terminus (13). The different sensitivity to IAM of the two enzymes when pretreated with cystine or Cys-803° implies teleologically that one possible role of acetylation at the N-terminus is protection of the native enzyme against physiological alkylation inactivation. Isoelectric Focusipg Analysip-m The inhibition studies described above could not reveal what kinds of enzyme-substrate intermediates were formed. We have already demonstrated that the exchange of Cys22 with Ser caused a protein pl shift (12). The wild-type enzyme ETT, mutant ETT-Ser's 7' n and mutant ETT-Ser -Ser were differentially treated with DTT, HED, cystine, Cys-803', and IAM as described in Experimental Procedures and analyzed by an isoelectric focusing gel, and the resulting pI values were examined (Fig. 27). Lanes 2 to 8 were the wild-type enzyme, ETT, treated with DTT, HED, cystine, Cys-$03., cystine/GSH, Cys-SOi/GSH, and IAM, respectively; lanes 9 to 15 were mutant ETT-Serz' incubated with DTT, HED, cystine, Cys—803', Cys-803' /GSH, IAM, and IAA, respectively; and lanes 16 to 19 were loaded with ”-Ser”, cystine, Cys-803°, Cys-SOi/GSH, and IAM treated mutant ETT-Ser respectively. Usually, when treated with DTT or HED, the enzyme is in the reduced (thiol) or oxidized (intramolecular disulfide) form, respectively, and their pI values are widely different from each other (Fig. 27, lane 2 and 3, lane 9 and 10). However, when treated with cystine or Cys-$03., the pI values of ETT and ETT-Ser's lay between those of their reduced and oxidized forms, i.e., the enzymes were neither in their thiol nor intramolecular disulfide forms, but rather, they may have been in partially mixed disulfide forms. In addition, more 170 Fig. 27 Isoelectric focusing apalysis of the ES intermediates of thioltrgnsferase. The wild-type enzyme, ETT, the mutants ETT--Ser25 12 , and ETT-Ser -Ser'2 were separately treated with DTT, HED, cystine, Cys-803', Cys—SOi/GSH, cystine/GSH, and 1AM as indicated with + (added) and - (not added). The pl value for each of the enzymes (either in the free state or in the modified state) was measured on a Servalyt Precoat isoelectric focusing gel, according to the manufacturer's instructions. The sample in each lane is indicated. 171 9.8.4.845 8.84.5. tm _.| filli- “|| u -. tire: . .. ---HOnHm Ill . u‘ 0““ II 112 '5 1" .25 ‘1. Ion.“ I+ll|l+ lllll ++Illl :mo I++lll++|ll|+l+111m09go Ill+Illl+ll|l+l+llee=a8 lllllllll +|lllll+l om: llllllllll+l lllll + to 1 rec at ons. 172 then one band existed in Cys-803' treated samples (lane 5 and lane 12), suggesting that both substrate components can form mixed disulfides with ETT and ETT-Ser's. The mixed disulfide formed between ETT and GSH was also observed in the presence of disulfide substrates and GSH, and the pl was slightly different from that of the enzyme-half—disulfide substrate form. The pls of IAM treated enzymes were quite similar to those of oxidized ones. This gave additional evidence that IAM reacted 2' eliminating its negative charge. For mutant, ETT-Sern-Ser'z, with Cys the pl values were equal to that of the oxidized form when treated with cystine, Cys—803', Cys-SO;°/GSH, and IAM. Thus, this mutant seems to favor the intramolecular disulfide form. Egdiogctive Labeling Studies—- For further testing of the mixed disulfides between the enzyme and its substrates, two radioactive labeled substrates, ["Clcystine and [IlllGSH, were used to track the reaction progress. The details of the labeling experiments were described in Experimental Procedures and the results are listed in Table VII. As shown in Table VII, enzyme-substrate intermediates were detected, since radioactivity was measured in the collected protein fractions both before and after the concentration of the enzymes. The mutant ETT- S er's [11 had the highest specific radioactivity (cpm/pg) both in C]cystine labeled samples or in ['H]GSH labeled samples. No counts 22 were detected in mutant ETT-Ser due to the absence of the active site 2'. When labeled by ["C]cystine, there were no counts detected in 's-Ser'z. This result agreed with that of the inhibition Cys mutant ETT-Ser studies and suggested that an intramolecular disulfide was formed in 25 this mutant. Pretreating ETT-Ser with IAM blocked ["C]cystine labeling. This is additional evidence for the identification of Cyszz as Table VII Radioactive labeling of thioltransferases The wild-type and mutant pig liver thioltransferases were incubated with ["C-]cystine, [glycine-Z-HIIGSH, or Cys-SO3./['H-]GSH in the presence of 100 mM sodium phosphate buffer, pH 7.5 for 20 min at room temperature, the excess radioactive labeled substrates were removed by Sephadex G-25, and the samples were concentrated under conditions described in the text. counted by liquid scintillation spectrometry and the specific radioactivity was calculated. The radioactivity of each enzyme was Substrates Thioltransferases -- 11 -- ---------------------- 3 -------- (0.3 mM) p—I C]cystine [inlcsn Cys-so' + [nlcsn (0.6 mM) (2.5 mM) (12.5 mM) (2.5 mM) cpm/ue cpm/ue Cpm/ue ETT 140 0 293 ETT-~3er22 o N.D.' 0 ETT-Serz' 386 0 2020 ETT-Ser's + IAMb o N.D. N.D. ETT-Val" 74 N.D. 114 ETT—Gln" 137 N.D. 242 ETT-Ser"-—Ser82 0 N.D. 83 a N.D. Not Determined b ETT-Serz' was pretreated with IAM, then with E—[U-"Clcysteine. 174 the active site, and alkylation at this position prevented labeling by either substrate. Except for the ETT-Sern mutant, all other tested enzymes were labeled by ['HlGSH in the presence of Cys-803'. Direct incubation of ETT with [°HlGSH, was unreactive, i.e., no isotope was incorporated into the enzyme. This result is also consistent with that of inhibition studies, i.e., GSH alone could not protect the enzyme against IAM inactivation. Thus, reduced enzyme must react with a disulfide substrate first, then with GSH to regenerate the dithiol or monothiol (ETT-Ser's) form. The wild-type enzyme, ETT, and the mutant ETT~-Ser25 treated with the radioactive labeled substrates as described above were subjected to SDS-PAGE under nonreducing conditions and the dried gel was subjected to autoradiography (Fig. 28). Lanes 1 to 3 were ETT treated with ["C]cystine, Cys-SOf/[xltllem and ['H]GSH, respectively, and lanes 4 25 to 7 were ETT-Ser treated with ["C]cystine, Cys-SO3'/['H]GSH, and IAM followed by either ["Clcystine, or ['Il]GSH, respectively. When treated with ["C] cystine or Cys-SOi/[ll-IIGSH, strong labeling was found in the mutant ETT-Serzs (Fig. 28, lane 4 and 5), indicating a covalent linkage between the enzyme and the substrate. With the same treatment, only faint signals were observed in ETT (Fig. 28, lanes 1 and 2) suggesting the formation of some intramolecular disulfide due to air oxidation during the electrophoresis process. No labeled bands could be seen when ETT--Ser25 was pretreated with IAM followed by ["C]cystine, or when either enzyme was directly incubated with [HilGSH (Fig. 28, lanes 3, 6 and 7). 175 Fig. 28 Autoradiography of ETT and ETT-Serz'. The wild-type (ETT) and mutant (ETT-Ser's) thioltransferases were treated with [11 radioactively labeled substrates, C]cystine or ['11]GSH, as described in Experimental Procedures. Lanes 1 to 3 were ETT treated with ["C]Cystine, Cys-SO'/['H]GSH, ['H-lGSH, respectively, lanes 4 to 7 were 3 ETT-Ser's treated with ["C]cystine, Cys-SO,°/[1H]GSH, and IAM followed by ["C]cystine, and ['H]GSH, respectively. 176 ETT ETT-ear25 fi I If I MW 1 2 3 4 5 6 7 (K) ,p, 11.7— )edifl 1 1 red” L 177 DISCUSSION Thiol-disulfide exchange reactions (Reaction 1) are actually nucleophilic ionic displacement reactions that take place either spontaneously or enzymatically, _i_r; vitro or 1:; vivo (15,16). Askeliif, et al. first suggested the name, thioltransferase, for the enzyme that catalyzed these reactions (4 and 5), and was originally called a transhydrogenase. This model proposed that two GSH molecules consecutively replace the disulfide substrate (RSSR) to yield two reduced products (2 RSH) and one GSSG molecule. A RSSG intermediate was formed during the catalytic reaction process when R was an aryl moiety, but not‘when cystine was the disulfide substrate (7). How the enzyme was involved in this reaction was not considered in this model. According to one model, the formation of the mixed disulfide between the enzyme and GSH is unlikely since GSH always replaces the enzyme to form RSSG and leaves the enzyme in the reduced form. E-S-S-R + GSH:R-S-S-G + E-s‘ + 14* (13) However, this model can not fully explain the data presented in this paper. For example, the disulfide between enzyme and GSH was demonstrated, and after preincubation with Cys-SOi/GSH, but not GSH, the enzyme was fully protected against IAM inhibition. In contrast, our results do support other models (Reactions 6 to 9 and 10 to 12). As shown in the inhibition studies, IAM can inactivate the reduced enzyme by reaction with the ionized sulfhydryl group of 22 Cys , but this inhibition can be variably prevented due to the formation of mixed~ or intramolecular—disulfides after pretreating the enzymes with 178 disulfide substrates, but not the mutants without Cysz'. Preincubation of the enzymes with Cys-SOi/GSH yielded much stronger protection. In addition, the enzymes could be labeled by ['H-IGSH in the presence of Cys-803. providing credence to Reactions 6-9 or 10—12. When treated with GSH or [SH-lGSH alone, the enzymes could not be protected against [AM or be radioactively labeled. These data were consistent with the sequence of the reactions in the second model, i.e., disulfide substrate adds first to the reduced enzyme, then GSH. The isoelectric focusing data are hard to explain according to Reaction 7. If an intramolecular disulfide is formed, the pl of the enzyme should be more alkaline and the enzyme would not be sensitive to inactivation by IAM. When treated with cystine, the wild-type enzyme (ETT) had a pl different from either the reduced form (treated with DTT) or oxidized form (treated with HED) and there are two such pl forms for the Cys-SO,- treated enzyme (Fig. 27). One form, representing 22 and cysteine, and the other may 22 a mixed disulfide between enzyme Cys represent a S-thiol sulfate ester between Cys 21 and -SO{ derived from the substrate. ETT as well as ETT-Gln is still partially inhibited by 2' with IAM after the above treatments. In addition, exchange of Cys Ser25 caused a 10% increase in thiol-disulfide exchange activity (11) and a 94% increase in DHA reductase activity (12). As an explanation, these results required an alternative mechanism from that shown by Reactions 7 and 8. For mutant, ETT-~Ser'a 22 -Ser , an intramolecular disulfide is formed, when treated with disulfide substrates. It has the same pl as that of the HED oxidized form and is not sensitive to IAM. Thus, we believe that, during the catalytic reactions, the formation of an intramolecular disulfide is an optional step dependent on the substrate E Tiff-i. ‘1."7: 2.1-.1115 , 179 involved. In the absence of GSH, the enzyme catalyzes Reaction 6, and whether Reaction 7 proceeds or not depends on the strength of the S- 25 and the thiol of the first nucleophilicity of the sulfhydryl group of Cys product, RSH (16). If the former is stronger, an intramolecular disulfide is formed (Reaction 7), whereas if the latter is stronger, the reaction will be equilibrated as in Reaction 6. In the presence of GSH, the reaction quickly proceeds to Reaction 8 directly from Reaction 6 or 7, then moves to Reaction 9 to complete a cycle. According to various results from this study and the considerations above, we suggested an alternative model (Reactions 10-12). This model represents a typical hexa—Uni Ping Pong mechanism, whereas the four step model (Reactions 6—9) is a Uni BiBiUni mechanism (17). The modified model may explain '5 is more efficient why the reaction catalyzed by the mutant ETT-Ser than that catalyzed by the wild-type enzyme. The mechanism of the DHA reductase activity of thioltransferase is not established. However, Cysn is likely to be the active site for both intrinsic enzymatic activities, and the mechanism of DHA reductase activity is presumably similar to that of a thiol-disulfide exchange activity, i.e., a thio-hemiketal intermediate, instead of a mixed disulfide, followed by displacement with GSH (18). Acknowledgment-- We thank Drs. Thomas L. Deits and Robert P. llausinger for helpful discussions, and Carol Smith for typing the manuscript. 2. 4. 9. 10. 11. 12. 13. 14. 15. 180 REFERENCES Racker, E. (1955) J. Biol. Chem. _2__1_’_1_, 867-874 Mannervik, B. (1986) in Thioredoxin and Glutaredoxin System: Structure and function (Holmgren, A., Branden, C.-T., Jtirnvall, H., and Sjoberg, B. eds.), pp 349-385, Raven Press, New York Fuchs, J. (1989) in Glutathione: Chemical, Biochemical, and Medical Aspect (Dolphin, D., Poulson, R., and Avramovic, O. eds.) Part B, pp 551-570, John Wiley 21 Sons, New York Hopper, S., Johnson, R. S., Vath, J. E., and Biemann, K. (1989) J. Biol. Chem. 2_6_4_, 20438-20447 Mannervik, B., and Axelsson, K. (1980) Biochem. J. fit), 125—130 Mannervik, B. (1980) in Enzymatic Basis of Detoxication (Jakoby, W. B. ed.) Vol 2, pp 229-244, Academic Press, New York Askeltif, P., Axelsson, K., Eriksson, S., and Mannervik, B. (1974) FEBS Lett. 3E, 263-267 Gan, Z.-R., and Wells, W. W. (1987) J. Biol. Chem. 2312, 6704-6707 Lindley, H. (1960) Biochem. J. 7_4, 577-584 Creighton, 'r. a. (1978) Prog. Biophys. Mol. Biol. a3, 231-297 Yang, Y., and Wells. W. W. (1991) J. Biol. Chem. (Submitted for publication, Chapter IV) Yang, Y., and Wells, W. W. (1991) J. Biol. Chem. (Submitted for publication, Chapter V) Yang, Y., and Wells, W. W. (1990), J. Biol. Chem. 2E5, 589-593 Gan, Z.-R. and Wells, W.W. (1987) Anal. Biochem. L624 265-273 Kosower, E. M. (1989) in Glutathione: Chemical, Biochemical, and Medical Aspect (Dolphin, D., Poulson, R., and Avramovic, O. eds.) Part A, pp 104-146 181 16. Parker, A.J. and Kaharasch, N. (1959) Chem. Rev. 51, 583-628 17. Cleland, W.W. (1963) Biochim. Biophy. Acta 51, 104-137 18. Lewin, S. (1976) in Vitamin C: Its Molecular Biology and Medical Potential, p. 38, Academic Press, New York, NY 182 CONCLUSIONS I started this dissertation by screening a pig liver cDNA library using polyclonal antibodies against pig liver thioltransferase (PLTT). A positive clone was sequenced and gave a deduced amino acid sequence identical to the directly determined one, except the N-terminus should be N-acetylalanine followed by a glutamine rather than the reverse as previously reported. The cDNA for PLTT was expressed at a high-level in E. 9911 JM105 in a soluble unfused active state. The establishment of the efficient PLTT expression system made it is possible to study the active site and catalytic mechanism of thioltransferase at a molecular level. The replacements of selected amino acids at the active center of PLTT by site-directed mutagenesis techniques directly confirmed that 22 is the catalytic site for this enzyme. The mutation studies also 22 Cys revealed that the extremely low pK. of the Cys (pK‘=3.8) is facilitated by Arg”. The mutants Ser sulfhydryl group 2" and Alaz‘l' are totally different in enzyme activity, the former has 110% of the wild type enzyme activity, whereas the latter shows only 9% activity remained. This result established that an amino acid residue with a more hydrophilic side chain e.g., -CHzOH or -CHz-SH, at position 25 is essential for optimal enzyme activity. The finding of the increased activity of the 25 22 mutant Ser implied that the intramolecular disulfide bond between Cys and Cysz' is not necessary at least for this mutant enzyme catalytic mechanism, and led to the suggestion of an alternative three step catalytic pathway. This dissertation also directly demonstrated that the 12 22 extra pair of cysteines, Cys and Cys , down-stream are not directly 183 involved in the enzyme catalytic action, but may have a structural function. The role of Lysz' was proposed to involve the stabilization the enzyme-substrate intermediate. The success of cloning, sequencing and expression of PLTT opened a wider door for extending studies of its structure and function. Sufficient recombinant wild type enzyme has been made in mg quantities, and its crystallization and three-dimensional structure is under investigation in collaboration with Dr. Suresh K. Katti, Yale University. Similar studies using mutant enzymes will be carried out in the near future. Unquestionably, future results from high resolution X-ray analysis will be very helpful in answering remaining critical questions related to the structure and mechanism of thioltransferase. The mutant enzymes will also be used to survey the possible catalytic mechanism of dehydroascorbate reductase activity. The cloned cDNA will be useful as follows: (1) as a probe to isolate the genomic DNA by screening a pig liver genomic DNA library, and sequencing the genomic DNA will reveal the splicing pattern of the thioltransferase gene; (2) to explore the thioltransferase distribution in tissues by Northern blot analysis; (3)in 111 yiyp studies of the function of this enzyme by expressing it in transformed mammalian cell line and observing potential changes in transfected cells, such as in drug resistance; (4) as a probe to screen other mammalian cDNAs, e.g., human placenta and rat liver for sequence analysis and future transfection studies in tissue culture.