Kinetics of irreversible inhibition of yeast alcohol dehydrogenase during modification by 4,4′-dithiodipyridine

The course of inactivation of yeast alcohol dehydrogenase (YADH) using 4,4′-dithiodipyridine (DSDP) has been studied in this paper. The results show that the reaction mechanism between DSDP and YADH is a competitive, complexing inhibition. The microscopic constants for the inactivation of the free enzyme and the enzyme-substrate complex were determined. The presence of the substrate NAD⁺ offers strong protection for this enzyme against inactivation by DSDP. The above results suggest that two Cys residues are essential for activity and are situated at the active site. These essential Cys residues should be Cys-46 and Cys-174 which are ligands to the catalytic zinc ion. Another Cys residue, which can be modified by DSDP, is non-essential for activity of the enzyme.     

Quinoprotein ethanol dehydrogenase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>: the unusual disulfide ring formed by adjacent cysteine residues is essential for efficient electron transfer to cytochrome <Emphasis Type="Italic">c</Emphasis><Subscript>550</Subscript>

All pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases contain an unusual disulfide ring formed between adjacent cysteine residues. A mutant enzyme that is lacking this structure was generated by replacing Cys105 and Cys106 with Ala in quinoprotein ethanol dehydrogenase (QEDH) from Pseudomonas aeruginosa ATCC17933. Heterologously expressed quinoprotein ethanol dehydrogenase in which Cys-105 and Cys-106 have been replaced by Ala (Cys105Ala/Cys106Ala apo-QEDH) was successfully converted to enzymatic active holo-enzyme by incorporation of its cofactor PQQ in the presence of Ca²⁺. The enzymatic activity of the mutant enzyme in the artificial dye test with N-methylphenazonium methyl sulfate (PMS) and 2,6-dichlorophenol indophenol (DCPIP) at pH 9 did not depend on an activating amine which is essential for wild type activity under these conditions. The mutant enzyme showed increased Michaelis constants for primary alcohols, while the affinity for the secondary alcohol 2-propanol was unaltered. Surprisingly, for all substrates tested the specific activity of the mutant enzyme in the artificial dye test was higher than that found for wild type QEDH. On the contrary, in the ferricyanide test with the natural electron acceptor cytochrome c ₅₅₀ the activity of mutant Cys105Ala/Cys106Ala was 15-fold lower than that of wild type QEDH. We demonstrate for the first time unambiguously that the unusual disulfide ring is essential for efficient electron transfer at pH 7 from QEDH to its natural electron acceptor cytochrome c ₅₅₀.     

Functioning of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Pma1 H<Superscript>+</Superscript>-ATPase carrying the minimal number of cysteine residues

Pma1 H⁺-ATPase is the primary proton pump in the plasma membrane of the yeast Saccharomyces cerevisiae. It generates an electrochemical proton gradient, thus providing energy for secondary solute transport systems. The enzyme contains nine cysteines, three (Cys148, Cys312, and Cys867) in transmembrane segments and the rest (Cys221, Cys376, Cys409, Cys472, Cys532, and Cys569) in the cytosolic parts of the molecule. Although individually they are not essential for the functioning of the ATPase, substitution of all of them leads to the loss of enzyme activity and impairment of biogenesis. By means of site-directed mutagenesis combined with other molecular-biological and biochemical methods, this work defines different combinations of minimal cysteine content that are consistent with ATPase function.     

Site-directed mutagenesis and chemical modification of the six native cysteine residues of the rat mitochondrial carnitine carrier: implications for the role of cysteine-136

By use of site-directed mutagenesis in combination with chemical modification of mutated proteins, the role of the six Cys residues in the transport function of the rat mitochondrial carnitine carrier (CAC) was studied. Several CAC mutants, in which one or more Cys residues had been replaced with Ser, were overexpressed in Escherichia coli, purified, and reconstituted in liposomes. The efficiency of incorporation into liposomes of the reconstituted proteins was lower for all constructs lacking Cys-23. Single, double, and quadruple replacement mutants showed V(max) comparable to that of the wild type. On the basis of the values of internal and external transport affinities (K(m)) for carnitine and of their comparison with those measured in mitochondria, the recombinant CAC is oriented unidirectionally in the liposomes, right side out compared to mitochondria. Substitution of Cys-136 with Ser caused a nearly complete loss of sensitivity of the CAC to N-ethylmaleimide, (2-aminoethyl)methanethiosulfonate hydrobromide (MTSES), and other hydrophilic SH reagents but not to the very hydrophobic N-phenylmaleimide. The wild-type CAC and the mutants containing Cys-136 showed substrate protection against NEM and MTSES inhibition and against NEM labeling. The data show that none of the native cysteines is essential for the transport mechanism and that Cys-136 is the major target of SH reagents and raise the hypothesis that Cys-136 is accessible from the external medium and is located at, or near, the substrate binding site. A model of the CAC is proposed in which the matrix hydrophilic loop containing Cys-136 protrudes into the membrane between the transmembrane domains of the protein.     

Novel insight into the mechanism of the vitamin K oxidoreductase (VKOR): electron relay through Cys43 and Cys51 reduces VKOR to allow vitamin K reduction and facilitation of vitamin K-dependent protein carboxylation

The vitamin K oxidoreductase (VKOR) reduces vitamin K to support the carboxylation and consequent activation of vitamin K-dependent proteins, but the mechanism of reduction is poorly understood. VKOR is an integral membrane protein that reduces vitamin K using membrane-embedded thiols (Cys-132 and Cys-135), which become oxidized with concomitant VKOR inactivation. VKOR is subsequently reactivated by an unknown redox protein that is currently thought to act directly on the Cys132-Cys135 residues. However, VKOR contains evolutionarily conserved Cys residues (Cys-43 and Cys-51) that reside in a loop outside of the membrane, raising the question of whether they mediate electron transfer from a redox protein to Cys-132/Cys-135. To assess a possible role, the activities of mutants with Ala substituted for Cys (C43A and C51A) were analyzed in intact membranes using reductants that were either membrane-permeable or -impermeable. Both reductants resulted in wild type VKOR reduction of vitamin K epoxide; however, the C43A and C51A mutants only showed activity with the membrane-permeant reductant. We obtained similar results when testing the ability of wild type and mutant VKORs to support carboxylation, using intact membranes from cells coexpressing VKOR and carboxylase. These results indicate a role for Cys-43 and Cys-51 in catalysis, suggesting a relay mechanism in which a redox protein transfers electrons to these loop residues, which in turn reduce the membrane-embedded Cys132-Cys135 disulfide bond to activate VKOR. The results have implications for the mechanism of warfarin resistance, the topology of VKOR in the membrane, and the interaction of VKOR with the carboxylase.     

Elimination of a reactive thiol group from the active site of chloramphenicol acetyltransferase.

1. The type III variant of chloramphenicol acetyltransferase (CATIII) is resistant to inactivation by ionizable modifying reagents such as 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and iodoacetate, whereas it is sensitive to inhibition by similar but uncharged reagents, including 4,4'-dithiodipyridine, methyl methanethiolsulphonate (MMTS) and iodoacetamide. The target for these thiol-modifying reagents has been postulated to be Cys-31. This residue is situated within a part of the chloramphenicol-binding site formed largely from the side chains of hydrophobic amino acid residues, which might be expected to discriminate against the access of ionized ligands to Cys-31. 2. The substitution of Cys-31 by alanine, serine, threonine or methionine yields an enzyme that is resistant to inactivation by thiol-specific reagents. Replacement of Cys-31 by alanine, serine or threonine results in increased Km values for chloramphenicol with only small changes in kcat.. In contrast, the Cys-31----Met substitution mainly affects kcat. values. Although the kcat. for chloramphenicol acetylation is decreased 13-fold compared with wild-type CAT, the kcat. for the acetyl-CoA hydrolysis reaction, which occurs in the absence of chloramphenicol, is increased 2.7-fold. 3. MMTS modification of cysteine residues results in an adduct (-CH2-S-S-CH3) that is structurally similar to the side chain of a methionine residue (-CH2-CH2-S-CH3). The kinetic properties of MMTS-modified CATIII closely resemble those of [Met31]CAT.     

Thermostabilization of recombinant human and bovine CuZn superoxide dismutases by replacement of free cysteines

Human CuZn superoxide dismutase (HSOD) has two free cysteines: a buried cysteine (Cys6) located in a beta-strand, and a solvent accessible cysteine (Cys111) located in a loop region. The highly homologous bovine enzyme (BSOD) has a single buried Cys6 residue. Cys6 residues in HSOD and BSOD were replaced by alanine and Cys111 residues in HSOD by serine. The mutant enzymes were expressed and purified from yeast and had normal specific activities. The relative resistance of the purified proteins to irreversible inactivation of enzymatic activity by heating at 70 degrees C was HSOD Ala6 Ser111 greater than BSOD Ala6 Ser109 greater than BSOD Cys6 Ser109 (wild type) greater than HSOD Ala6 Cys111 greater than HSOD Cys6 Ser111 greater than HSOD Cys111 (wild type). In all cases, removal of a free cysteine residue increased thermostability.     

Use of a disulfide cross-linking strategy to study muscarinic receptor structure and mechanisms of activation.

To gain insight into the molecular architecture of the cytoplasmic surface of G protein-coupled receptors, we have developed a disulfide cross-linking strategy using the m3 muscarinic receptor as a model system. To facilitate the interpretation of disulfide cross-linking data, we initially generated a mutant m3 muscarinic receptor (referred to as m3'(3C)-Xa) in which most native Cys residues had been deleted or substituted with Ala or Ser (remaining Cys residues Cys-140, Cys-220, and Cys-532) and in which the central portion of the third intracellular loop had been replaced with a factor Xa cleavage site. Radioligand binding and second messenger assays showed that the m3'(3C)-Xa mutant receptor was fully functional. In the next step, pairs of Cys residues were reintroduced into the m3'(3C)-Xa construct, thus generating 10 double Cys mutant receptors. All 10 mutant receptors contained a Cys residue at position 169 at the beginning of the second intracellular loop and a second Cys within the C-terminal portion of the third intracellular loop, at positions 484-493. Radioligand binding studies and phosphatidylinositol assays indicated that all double Cys mutant receptors were properly folded. Membrane lysates prepared from COS-7 cells transfected with the different mutant receptor constructs were incubated with factor Xa protease and the oxidizing agent Cu(II)-(1,10-phenanthroline)3, and the formation of intramolecular disulfide bonds between juxtaposed Cys residues was monitored by using a combined immunoprecipitation/immunoblotting strategy. To our surprise, efficient disulfide cross-linking was observed with 8 of the 10 double Cys mutant receptors studied (Cys-169/Cys-484 to Cys-491), suggesting that the intracellular m3 receptor surface is characterized by pronounced backbone fluctuations. Moreover, [35S]guanosine 5'-3-O-(thio)triphosphate binding assays indicated that the formation of intramolecular disulfide cross-links prevented or strongly inhibited receptor-mediated G protein activation, suggesting that the highly dynamic character of the cytoplasmic receptor surface is a prerequisite for efficient receptor-G protein interactions. This is the first study using a disulfide mapping strategy to examine the three-dimensional structure of a hormone-activated G protein-coupled receptor.     

Role of cysteine residues in human NADH-cytochrome b5 reductase studied by site-directed mutagenesis. Cys-273 and Cys-283 are located close to the NADH-binding site but are not catalytically essential

Human NADH-cytochrome b5 reductase (EC 1.6.2.2) contains 4 cyteine residues (Cys-203, -273, -283, and -297). Cys-283 was previously proposed to be involved in NADH binding by chemical modification (Hackett, C. S., Novoa, W. B., Ozols, J., and Strittmatter, P. (1986) J. Biol. Chem. 261, 9854-9857). In the present study the role of cysteines in the enzyme was probed by replacing these residues by Ser, Ala, or Gly employing site-directed mutagenesis and chemical modification. Four mutants, in which 1 of the 4 Cys residues was replaced by Ser, retained comparable kcat and Km values to those of the wild type. All of these mutants were as sensitive as the wild type to treatment with SH modifiers, while a double mutant, C273S/C283S was resistant. Since inhibition by SH modifiers was protected by NADH, Cys-273 and Cys-283 were implicated to be close to the NADH-binding site. C273A and C273A/C283A mutants showed approximately one-fifth of the enzyme-FAD reduction rate of the wild type as revealed by steady-state kinetics and by stopped-flow analysis. Anaerobic titration has shown that reduction and re-oxidation processes including formation of the red semiquinone of these mutants were not significantly altered from those of the wild type. From these results it was concluded that none of the Cys residues of the enzyme are essential in the catalytic reaction, but Cys-273 conserved among the enzymes homologous to NADH-cytochrome b5 reductase homologous to NADH-cytochrome b5 reductase plays role(s) in facilitating the reaction. A difference spectrum with a peak at 317 nm, which was formerly considered to be derived from the interaction between NAD+ and Cys-283 of the reduced enzyme, appeared upon binding of NAD+ not only to the reduced wild type enzyme but also to the C273A/C283A mutant in which both of the Cys residues close to the NADH-binding site were replaced.     

Role of cysteine residues in glutathione synthetase from Escherichia coli B. Chemical modification and oligonucleotide site-directed mutagenesis

Escherichia coli B glutathione synthetase is composed of four identical subunits; each subunit contains 4 cysteine residues (Cys-122, -195, -222, and -289). We constructed seven different mutant enzymes containing 3, 2, or no cysteine residues/subunit by replacement of cysteine codons with those of alanine in the gsh II gene using site-directed mutagenesis. Three mutant enzymes, Ala289, Ala222/289, Cys-free (Ala122/195/222/289), in which cysteine at residue 289 was replaced with alanine, were not inactivated by 5,5'-dithiobis(2-nitrobenzoate) (DTNB), while the other four mutants retaining Cys-289 were inactivated at the wild-type rate. From these selective inactivations of mutant enzymes by DTNB, the sulfhydryl group modified by DTNB was unambiguously identified as Cys-289. In this way, Cys-289 was found to be also a target of modification with 2-nitrothiocyanobenzoate and N-ethylmaleimide, while Cys-195 was of p-chloromercuribenzoate. These results suggest that both Cys-195 and Cys-289 were not essential for the activity of the glutathione synthetase, but chemical modification of either one of the two sulfhydryl groups resulted in complete loss of the activity. Replacement of Cys-122 to Ala-122 enhanced the reactivity of Cys-289 with sulfhydryl reagents.     

Metal-tetracycline/H+ antiporter of Escherichia coli encoded by a transposon, Tn10. The role of the conserved dipeptide, Ser65-Asp66, in tetracycline transport

The transposon Tn10-encoded tetracycline resistance protein functions as a metal-tetracycline/H+ antiporter (Yamaguchi, A., Udagawa, T., and Sawai, T. (1990) J. Biol. Chem. 265, 4809-4813). The Ser65-Asp66 dipeptide is conserved in all known tetracycline antiporter proteins and is an important target for site-directed mutagenesis. When Asp66 was replaced by Asn, the transport activity was completely lost, whereas when it was replaced by Glu, the activity was reduced to 10% of the wild-type level, indicating that a negative charge at position 66 is essential for tetracycline transport. Replacement of Ser65 by Cys or Ala, in contrast, caused only a minor change in tetracycline transport activity. However, the Cys65 mutant antiporter was sensitive to sulfhydryl reagents. Complete inactivation of the Cys65 antiporter by N-ethylmaleimide was not prevented by the substrate. A less bulky reagent, methyl methanethiosulfonate, caused partial inactivation of the Cys65 antiporter without changing its affinity to the substrate. These results indicate that a region including the dipeptide plays an important role in metal-tetracycline transport except for substrate binding. It may act as a gate which opens on the charge-charge interaction between Asp66 and the metal-tetracycline.     

Effect of cysteine residues on the activity of arginyl-tRNA synthetase from Escherichia coli.

Arginyl-tRNA synthetase (ArgRS) from Escherichia coli (E. coli) contains four cysteine residues. In this study, the role of cysteine residues in the enzyme has been investigated by chemical modification and site-directed mutagenesis. Titration of sulfhydryl groups in ArgRS by 5, 5'-dithiobis(2-nitro benzoic acid) (DTNB) suggested that a disulfide bond was not formed in the enzyme and that, in the native condition, two DTNB-sensitive cysteine residues were located on the surface of ArgRS, while the other two were buried inside. Chemical modification of the native enzyme by iodoacetamide (IAA) affected only one DTNB-sensitive cysteine residue and resulted in 50% loss of enzyme activity, while modification by N-ethylmeimide (NEM) affected two DTNB-sensitive residues and caused a complete loss of activity. These results, when combined with substrate protection experiments, suggested that at least the two cysteine residues located on the surface of the molecule were directly involved in substrates binding and catalysis. However, changing Cys to Ala only resulted in slight loss of enzymatic activity and substrate binding, suggesting that these four cysteine residues in E. coli ArgRS were not essential to the enzymatic activity. Moreover, modifications of the mutant enzymes indicated that the two DTNB- and NEM-sensitive residues were Cys(320) and Cys(537) and the IAA-sensitive was Cys(320). Our study suggested that inactivation of E. coli ArgRS by sulfhydryl reagents is a result of steric hindrance in the enzyme.     

Cysteine residues are not essential for the catalytic activity of human class Mu glutathione transferase M1a-1a

To investigate the possible involvement of a Cys thiol in the catalysis of the human glutathione transferase M1a-1a, we constructed mutants of this enzyme wherein the four Cys residues present in the native enzyme were replaced by Ala residues. Three mutants, one where all four Cys residues had been replaced and two mutants where three out of four Cys residues were changed into Ala, were characterized regarding their catalytic activities with three different substrates as well as by their binding of three different inhibitors. All three Cys-deficient mutant forms of glutathione transferase M1a-1a were catalytically active with the tested substrates and their binding of inhibitors, measured by I50, were not significantly different from the values previously obtained for the wild-type enzyme. We therefore conclude that none of the Cys residues in this class Mu glutathione transferase are directly involved in the catalysis performed by this enzyme.     

Importance of Cys,Gln, and Tyr from the transmembrane domain of human alpha 3/4 fucosyltransferase III for its localization and sorting in the Golgi of baby hamster kidney cells

Human fucosyltransferase III (EC ) (FT3wt) is localized in the Golgi of baby hamster kidney cells and synthesizes Lewis determinants associated with cell adhesion events. Replacement of the amino acid residues from the transmembrane domain (TM) Cys-16, Gln-23, Cys-29, and Tyr-33 by Leu (FT3np) caused a shift in enzyme localization to the plasma membrane. The mislocalization caused a dramatic decrease in the amount of biosynthetic products of FT3wt, the Lewis determinants. Determination of the expression levels on the surface with mutants of the enzyme, where one, two, or three of these residues were replaced by Leu, suggested that Cys from the TM was required for the localization of FT3 in the Golgi. Furthermore, Cys-23 and Cys-29 mediated the formation of disulfide-bonded dimers but not higher molecular weight oligomers. In vitro reconstitution of intra-Golgi transport showed that FT3wt was incorporated into coatomer protein (COP) I vesicles, contrary to FT3np. These data suggested that Cys, Gln, and Tyr residues are important for FT3wt sorting into the transport vesicles possibly due to interactions with other membrane proteins.     

Mapping of reactive sulfhydryls in avian liver 3-hydroxy-3-methylglutaryl coenzyme A synthase

Avian liver mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase contains seven sulfhydryls per 53 kDa subunit. Peptides that harbor these sulfhydryls can be mapped by reverse-phase HPLC separation of tryptic digests of denatured 14C-carboxymethylated enzyme. Native enzyme is inactivated by a variety of reagents that target cysteine residues. Of particular interest is the enzyme's sensitivity to reagents (e.g., CdCl2, copper phenanthroline) that target vicinal thiols. The identity of the cysteines which are modified by these reagents can be determined by peptide mapping after denaturation. 14C-carboxymethylation and trypsin digestion of the sample. While the extent of reaction of any particular cysteinyl sulfhydryl depends on the identity of the reagent employed, three of the protein's seven cysteinyl sulfhydryls are frequently modified upon inactivation of the enzyme. The peptides which contain these reactive sulfhydryls have been isolated and their sequences have been determined by Edman degradation techniques. Comparison of these sequences with the deduced primary structure of the rodent cytosolic enzyme (Gil et al. (1986) J. Biol. Chem. 261, 3710) indicates strong homologies. These homologies allow assignment of the reactive residues as Cys-129, Cys-224 and Cys-268. The sensitivity of these residues to reagents that target vicinal thiols, coupled with the fact that cys-129 is the residue involved in formation of the acyl-S-enzyme intermediate (Vollmer et al. (1988) Biochemistry 27, 4288), suggests that these three residues may be closely juxtaposed within the enzyme's catalytic domain.     

Alternating access and a pore-loop structure in the Na+-citrate transporter CitS of Klebsiella pneumoniae

CitS of Klebsiella pneumoniae is a secondary transporter that transports citrate in symport with 2 Na(+) ions. Reaction of Cys-398 and Cys-414, which are located in a cytoplasmic loop of the protein that is believed to be involved in catalysis, with thiol reagents resulted in significant inhibition of uptake activity. The reactivity of the two residues was determined in single Cys mutants in different catalytic states of the transporter and from both sides of the membrane. The single Cys mutants were shown to have the same transport stoichiometry as wild type CitS, but the C398S mutation was responsible for a 10-fold loss of affinity for Na(+). Both cysteine residues were accessible from the periplasmic as well as from the cytoplasmic side of the membrane by the membrane-impermeable thiol reagent [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET) suggesting that the residues are part of the translocation site. Binding of citrate to the outward facing binding site of the transporter resulted in partial protection against inactivation by N-ethylmaleimide, whereas binding to the inward facing binding site resulted in essentially complete protection. A 10-fold higher concentration of citrate was required at the cytoplasmic rather than at the periplasmic side of the membrane to promote protection. Only marginal effects of citrate binding were seen on reactivity with MTSET. Binding of Na(+) at the periplasmic side of the transporter protected both Cys-398 and Cys-414 against reaction with the thiol reagents, whereas binding at the cytoplasmic side was less effective and discriminated between Cys-398 and Cys-414. A model is presented in which part of the cytoplasmic loop containing Cys-398 and Cys-414 folds back into the translocation pore as a pore-loop structure. The loop protrudes into the pore beyond the citrate-binding site that is situated at the membrane-cytoplasm interface.     

Role of cysteine residues in 4-oxalomesaconate hydratase from Pseudomonas ochraceae NGJ1

4-Oxalomesaconate hydratase from Pseudomonas ochraceae NGJ1 is unstable in the absence of reducing reagents such as dithiothreitol, and strongly inhibited by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). To study the role of cysteine residues in enzyme catalysis, the eight individual cysteine residues of the enzyme were replaced with serine residues by site-directed mutagenesis. The catalytic properties and chemical modification of wild- and mutant type-enzymes by DTNB showed that (i) none of eight cysteine residues was essential for enzyme catalysis; (ii) the inhibition by DTNB was mostly due to modification of Cys-186; (iii) Cys-96 might be another residue reacting with DTNB, and its modification caused an increase in the K(m)-value for 4-oxalomesaconate; (iv) the other six cysteine residues were inaccessible to DTNB, but susceptible to HgCl(2); and (v) only replacement of Cys-186 remarkably improved the stability of the enzyme in the absence of reducing reagent.     

Cys-X scanning for expansion of active-site residues and modulation of catalytic functions in a glutathione transferase

We propose Cys-X scanning as a semisynthetic approach to engineer the functional properties of recombinant proteins. As in the case of Ala scanning, key residues in the primary structure are identified, and one of them is replaced by Cys via site-directed mutagenesis. The thiol of the residue introduced is subsequently modified by alternative chemical reagents to yield diverse Cys-X mutants of the protein. This chemical approach is orthogonal to Ala or Cys scanning and allows the expansion of the repertoire of amino acid side chains far beyond those present in natural proteins. In its present application, we have introduced Cys-X residues in human glutathione transferase (GST) M2-2, replacing Met-212 in the substrate-binding site. To achieve selectivity of the modifications, the Cys residues in the wild-type enzyme were replaced by Ala. A suite of simple substitutions resulted in a set of homologous Met derivatives ranging from normethionine to S-heptyl-cysteine. The chemical modifications were validated by HPLC and mass spectrometry. The derivatized mutant enzymes were assayed with alternative GST substrates representing diverse chemical reactions: aromatic substitution, epoxide opening, transnitrosylation, and addition to an ortho-quinone. The Cys substitutions had different effects on the alternative substrates and differentially enhanced or suppressed catalytic activities depending on both the Cys-X substitution and the substrate assayed. As a consequence, the enzyme specificity profile could be changed among the alternative substrates. The procedure lends itself to large-scale production of Cys-X modified protein variants.     

Fluorescence energy transfer studies of human deoxycytidine kinase: role of cysteine 185 in the conformational changes that occur upon substrate binding

Human deoxycytidine kinase (dCK) phosphorylates both pyrimidine and purine deoxynucleosides, including numerous nucleoside analogue prodrugs. Energy transfer studies of transfer between Trp residues of dCK and the fluorescent probe N-(1-pyrene)maleimide (PM), which specifically labels Cys residues in proteins, were performed. Two of the six Cys residues in dCK were labeled, yielding a protein that was functionally active. We determined the average distances between PM-labeled Cys residues and Trp residues in dCK in the absence and presence of various pyrimidine and purine nucleoside analogues with the Trp residues as energy donors and PM-labeled Cys residues as acceptors. The transfer efficiency was determined from donor intensity quenching and the F?rster distance R(0) at which the efficiency of energy transfer is 50%, which was 19.90 A for dCK-PM. The average distance R between the Trp residues and the labeled Cys residues in dCK-PM was 18.50 A, and once substrates bound, this distance was reduced, demonstrating conformational changes. Several of the Cys residues of dCK were mutated to Ala, and the properties of the purified mutant proteins were studied. PM labeled a single Cys residue in Cys-185-Ala dCK, suggesting that one of the two Cys residues labeled in wild-type dCK was Cys 185. The distance between the single PM-labeled Cys residue and the Trp residues in Cys-185-Ala dCK was 20.75 A. Binding of nucleosides had no effect on the pyrene fluorescence of Cys-185-Ala dCK, indicating that the conformational changes observed upon substrate binding to wild-type dCK-PM involved the "lid region" of which Cys 185 is a part. The substrate specificity of Cys-185-Ala dCK was altered in that dAdo and UTP were better substrates for the mutant than for the wild-type enzyme.     

The functional role of cysteines in isopenicillin N synthase. Correlation of cysteine reactivities toward sulfhydryl reagents with kinetic properties of cysteine mutants

Isopenicillin N synthase (IPNS) from Cephalosporium acremonium contains 2 cysteine residues in positions 106 and 255 which are invariant in all IPNS sequences reported to date (Miller, J.R., and Ingolia, T.D. (1989) Mol. Microbiol. 3, 689-695). Although these residues have been postulated to play a role in catalysis (Samson, S.M., Chapman, J.L., Belagaje, R., Queener, S., and Ingolia, T.D. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5705-5709) as well as enzyme inactivation (Perry, D., Abraham, E.P., and Baldwin, J.E. (1988) Biochem. J. 255, 345-351) little information exists regarding their oxidation state and reactivity. In this paper, the functions of these cysteines have been addressed by chemical modification techniques in combination with site-directed mutagenesis. In the intact wild type protein, both cysteines are inert toward 5,5'-dithiobis-(2-nitrobenzoic acid) and iodoacetic acid. However, Cys-106, but not Cys-255, can be slowly modified by N-ethylmaleimide, and its modification is partially blocked by the presence of a substrate analog inhibitor. Complete modification of both cysteines by sulfhydryl reagents requires unfolding of the protein but not the presence of a disulfide reducing agent. The thiol content of IPNS is shown to be the same before and after exposing the enzyme to substrate even though during catalysis the enzyme is rapidly inactivated. The impact on catalysis due to alteration of the cysteines has been assessed using three site-specific mutants: Cys-106----Ser, Cys-255----Ser, and Cys-106,255----Ser. These mutant proteins have been purified as apoenzymes with the nature of the mutation verified by peptide mapping. The stoichiometry of metal required for activity remains as one equivalent of Fe2+/mol of enzyme in the mutants as in wild type IPNS. Compared with wild type, Cys-255----Ser shows a reduction in Vmax by 33%, and an increase in Km by 1.4-fold, while Cys-106----Ser and Cys-106,255----Ser, which have identical kinetic properties, exhibit a decrease in Vmax by 63% but an elevation of Km by 14-fold. The data presented demonstrate that 1) both cysteines are free thiols; 2) Cys-106 is more exposed than Cys-255; 3) substrate-induced inactivation is not caused by cysteine modification; 4) neither cysteine is absolutely essential for bond making or breaking events during catalysis.(ABSTRACT TRUNCATED AT 400 WORDS)