Mechanistic consequences of mutation of active site carboxylates in a retaining beta-1,4-glycanase from Cellulomonas fimi

The exoglucanase/xylanase Cex from Cellulomonas fimi is a retaining glycosidase which functions via a two-step mechanism involving the formation and hydrolysis of a covalent glycosyl-enzyme intermediate. The roles of three conserved active site carboxylic acids in this enzyme have been probed by detailed kinetic analysis of mutants modified at these three positions. Elimination of the catalytic nucleophile (E233A) results in an essentially inactive enzyme, consistent with the important role of this residue. However addition of small anions such as azide or formate restores activity, but as an inverting enzyme since the product formed under these conditions is the alpha-glycosyl azide. Shortening of the catalytic nucleophile (E233D) reduces the rates of both formation and hydrolysis of the glycosyl-enzyme intermediate some 3000-4000-fold. Elimination of the acid/base catalyst (E127A) yields a mutant for which the deglycosylation step is slowed some 200-300-fold as a consequence of removal of general base catalysis, but with little effect on the transition state structure at the anomeric center. Effects on the glycosylation step due to removal of the acid catalyst depend on the aglycon leaving group ability, with minimal effects on substrates requiring no general acid catalysis but large (> 10(5)-fold) effects on substrates with poor leaving groups. The Br?nsted beta 1g value for hydrolysis of aryl cellobiosides was much larger (beta 1g approximately -1) for the mutant than for the wild-type enzyme (beta 1g = -0.3), consistent with removal of protonic assistance. The pH-dependence was also significantly perturbed. Mutation of a third conserved active site carboxylic acid (E123A) resulted in rate reductions of up to 1500-fold on poorer substrates, which could be largely restored by addition of azide, but without the formation of glycosyl azide products. These results suggest a simple strategy for the identification of the key active site nucleophile and acid/base catalyst residues in glycosidases without resort to active site labeling.     

Site-directed mutagenesis of active site contact residues in a hydrolytic abzyme: evidence for an essential histidine involved in transition state stabilization

Specific molecular interactions involved in catalysis by antibody 6D9 were investigated by site-directed mutagenesis. The catalytic antibody 6D9, which was generated against a transition state analog (III), hydrolyzes a non-bioactive chloramphenicol monoester derivative (I) to produce chloramphenicol (II). Construction of a three-dimensional molecular model of 6D9 and sequence comparison within a panel of related antibodies suggested candidates for catalytic residues, His (L27d), Tyr (L32), Tyr (H58) and Arg (H100b); these were targeted for the site-directed mutagenesis study. The Y-H58-F and R-H100b-A mutants possessed catalytic activities comparable to that of the wild-type, and the Y-H58-H and Y-L32-F mutant displayed an approximately fivefold decrease in k(cat)/Km. In the transition state analysis, the plots of logK(TSA) versus log(k(cat)/Km) for the mutants are linear, with a slope of approximately 1.0, indicating that the entire hapten-binding energy in the mutants is also utilized to bind the transition state and to accelerate the catalysis. In addition, a dramatic change in the catalytic activity was observed when the histidine residue (27d) in the CDR1 light chain was replaced with alanine. The H-L27d-A mutant had no detectable catalytic activity. This mutation led to a large, 40-fold reduction in transition state binding, with no change in substrate binding. Coupled with the previous kinetic studies and chemical modifications of the intact 6D9 antibody, this mutagenesis study has demonstrated that His L27d plays an essential role in stabilization of the transition state, the mechanism of catalysis by the 6D9 antibody.     

Sulfatases, trapping of the sulfated enzyme intermediate by substituting the active site formylglycine

Sulfatases contain an active site formylglycine residue that is generated by post-translational modification. Crystal structures of two lysosomal sulfatases revealed significant similarity to the catalytic site of alkaline phosphatase containing a serine at the position of formylglycine. To elucidate the catalytic mechanism of sulfate ester hydrolysis, the formylglycine of arylsulfatases A and B was substituted by serine. These mutants upon incubation with substrate were covalently sulfated at the introduced serine. This sulfated enzyme intermediate was stable at pH 5. At alkaline pH it was slowly hydrolyzed. These characteristics are analogous to that of alkaline phosphatase which forms a phosphoserine intermediate that is stable at pH 5, but is hydrolyzed at alkaline pH. In wild-type sulfatases the hydroxyl needed for formation of the sulfated enzyme intermediate is provided by the aldehyde hydrate of the formylglycine. The second, non-esterified hydroxyl of the aldehyde hydrate is essential for rapid desulfation of the enzyme at acidic pH, which most likely occurs by elimination. The lack of this second hydroxyl in the serine mutants explains the trapping of the sulfated enzyme intermediate. Thus, in acting as a geminal diol the formylglycine residue allows for efficient ester hydrolysis in an acidic milieu.     

Analysis of stress in the active site of myosin accompanied by conformational changes in transient state intermediate complexes using photoaffinity labeling and 19F-NMR spectroscopy

Myosin forms stable ternary complexes with ADP and the phosphate analogues, fluoroaluminate (Al F4-), fluoroberyllate (BeFn) or orthovanadate (Vi); these ternary complexes mimic transient intermediates in the myosin ATPase cycle. Moreover, we previously demonstrated that these complexes may mimic different myosin ATPase reaction intermediates corresponding to separate steps in the cross-bridge cycle [Maruta, S., Henry, G. D., Sykes, B. D. & Ikebe, M. (1993) J. Biol. Chem. 268, 7093-7100]. Park et al. suggested that the changing conformation of ATP during hydrolysis stresses the active site of myosin subfragment-1 (S-1) through protein-nucleotide contacts at the gamma-phosphate and nucleotide base, and the stress-induced strain in the cross-bridge may be the mechanism by which energy in ATP is transferred to the myosin structure [Park, S., Ajtai, K. & Burghardt, T. P. (1997) Biochemistry 36, 3368-3372]. In the present study, the photoactive ADP analogue, 3'-O-(N-methylanthraniloyl)-2-azido-ADP (Mant-2-N3-ADP), and the 19F-labeled ADP analogue, 2-[(trifluoromethylnitrophenyl)aminoethyl]diphosphate, were employed to examine conformational differences in protein-nucleotide contact in the ATP-binding site that may correlate with energy transduction. Mant-2-N3-ADP was trapped within the active site of skeletal and smooth muscle myosin in the presence of AlF4-, BeFn or Vi. For both skeletal and smooth muscle myosins, trapped Mant-2-N3-ADP was covalently linked to the 25-kDa N-terminal fragment of S-1 of both myosin/Mant-2-N3-ADP/AlF4- and BeFn complexes, presumably at Trp130. However, the efficiency of the incorporation was much higher for skeletal than for smooth muscle myosin suggesting that the conformations of the adenine-binding pockets of the two myosins are somewhat different. Although the amount of Mant-2-N3-ADP trapped in the presence of AlF4- and BeFn was the same for both myosins, the efficiency of photolabeling skeletal muscle myosin was approximately two times higher for BeFn complex than for AlF4- complex. The 19F-NMR spectra of the bound 2-[(trifluoromethylnitrophenyl)aminoethyl]diphosphate in the ternary complexes formed in the presence of AlF4-, BeFn or Vi showed small but distinguishable differences. Taken together, these results indicate that there is some variation in the protein-nucleotide contacts at the nucleotide base among the ternary complexes studied, and these differences mimic separate steps occurring transiently during the contractile cycle.     

Active serine involved in the stabilization of the active site loop in the Humicola lanuginosa lipase

We have investigated the binding properties of and dynamics in Humicola lanuginosa lipase (Hll) and the inactive mutant S146A (active Ser146 substituted with Ala) using fluorescence spectroscopy and molecular dynamics simulations, respectively. Hll and S146A show significantly different binding behavior for phosphatidylcholine (PC) and phosphatidylglycerol (PG) liposomes. Generally, higher binding affinity is observed for Hll than the S146A mutant. Furthermore, depending on the matrix, the addition of the transition state analogue benzene boronic acid increases the binding affinity of S146A, whereas only small changes are observed for Hll suggesting that the active site lid in the latter opens more easily and hence more lipase molecules are bound to the liposomes. These observations are in agreement with molecular dynamics simulations and subsequent essential dynamics analyses. The results reveal that the hinges of the active site lid are more flexible in the wild-type Hll than in S146A. In contrast, larger fluctuations are observed in the middle region of the active site loop in S146A than in Hll. These findings reveal that the single mutation (S146A) of the active site serine leads to substantial conformational alterations in the H. lanuginosa lipase and different binding affinities.     

Contribution of cutinase serine 42 side chain to the stabilization of the oxyanion transition state

Cutinase from the fungus Fusarium solani pisi is a lipolytic enzyme able to hydrolyze both aggregated and soluble substrates. It therefore provides a powerful tool for probing the mechanisms underlying lipid hydrolysis. Lipolytic enzymes have a catalytic machinery similar to those present in serine proteinases. It is characterized by the triad Ser, His, and Asp (Glu) residues, by an oxyanion binding site that stabilizes the transition state via hydrogen bonds with two main chain amide groups, and possibly by other determinants. It has been suggested on the basis of a covalently bond inhibitor that the cutinase oxyanion hole may consist not only of two main chain amide groups but also of the Ser42 O gamma side chain. Among the esterases and the serine and the cysteine proteases, only Streptomyces scabies esterase, subtilisin, and papain, respectively, have a side chain residue which is involved in the oxyanion hole formation. The position of the cutinase Ser42 side chain is structurally conserved in Rhizomucor miehei lipase with Ser82 O gamma, in Rhizopus delemar lipase with Thr83 O gamma 1, and in Candida antartica B lipase with Thr40 O gamma 1. To evaluate the increase in the tetrahedral intermediate stability provided by Ser42 O gamma, we mutated Ser42 into Ala. Furthermore, since the proper orientation of Ser42 O gamma is directed by Asn84, we mutated Asn84 into Ala, Leu, Asp, and Trp, respectively, to investigate the contribution of this indirect interaction to the stabilization of the oxyanion hole. The S42A mutation resulted in a drastic decrease in the activity (450-fold) without significantly perturbing the three-dimensional structure. The N84A and N84L mutations had milder kinetic effects and did not disrupt the structure of the active site, whereas the N84W and N84D mutations abolished the enzymatic activity due to drastic steric and electrostatic effects, respectively.     

Mechanism of phosphorylation-recognition by visual arrestin and the transition of arrestin into a high affinity binding state

Arrestin plays an important role in quenching phototransduction via its ability to interact specifically with the phosphorylated light-activated form of the visual receptor rhodopsin (P-Rh*). Previous studies have demonstrated that Arg175 in bovine arrestin is directly involved in the phosphorylation-dependent binding of arrestin to rhodopsin and seems to function as a phosphorylation-sensitive trigger. In this study, we further probed the molecular mechanism of phosphorylation recognition by substituting 19 different amino acids for Arg175. We also assessed the effects of mutagenesis of several other highly conserved residues within the phosphorylation-recognition region (Val170, Leu172, Leu173, Ile174, Val177, and Gln178). The binding of all of these mutants to P-Rh*, light-activated rhodopsin, and truncated rhodopsin, which lacks the carboxyl-terminal phosphorylation sites, was then characterized. Overall, our results suggest that arrestin interaction with the phosphorylated carboxyl-terminal domain of rhodopsin activates two relatively independent changes in arrestin: (a) mobilization of additional binding sites and (b) increased affinity of the phosphorylation-recognition region for the rhodopsin carboxyl-terminal domain. Together, these two mechanisms ensure the exquisite selectivity of arrestin toward P-Rh*. Mutagenesis of residues that play a major role in binding site mobilization and phosphorylation-recognition enabled us to create "constitutively active" (phosphorylation-independent) arrestin mutants that have high affinity for both P-Rh* and light-activated rhodopsin. The introduction of a negative charge in position 175 was particularly effective in this respect. A detailed molecular model of phosphorylation-recognition is proposed.     

Stereochemical course of enzymatic enolpyruvyl transfer and catalytic conformation of the active site revealed by the crystal structure of the fluorinated analogue of the reaction tetrahedral intermediate bound to the active site of the C115A mutant of MurA

MurA (UDP-GlcNAc enolpyruvyl transferase), the first enzyme in bacterial peptidoglycan biosynthesis, catalyzes the enolpyruvyl transfer from phosphoenolpyruvate (PEP) to the 3'-OH of UDP-GlcNAc by an addition-elimination mechanism that proceeds through a tetrahedral ketal intermediate. The crystal structure of the Cys115-to-Ala (C115A) mutant of Escherichia coli MurA complexed with a fluoro analogue of the tetrahedral intermediate revealed the absolute configuration of the adduct and the stereochemical course of the reaction. The fluorinated adduct was generated in a preincubation of wild-type MurA with (Z)-3-fluorophosphoenolpyruvate (FPEP) and UDP-GlcNAc and purified after enzyme denaturation. The fluorine substituent stabilizes the tetrahedral intermediate toward decomposition by a factor of 10(4)-10(6), facilitating manipulation of the adduct. The C115A mutant of MurA was utilized to avoid the microheterogeneity that arises in the wild-type MurA from the attack of Cys115 on C-2 of FPEP in competition with the formation of the fluorinated adduct. The crystal structure of the complex was determined to 2.8 A resolution, and the absolute configuration at C-2 of the adduct was found to be 2R. Thus, addition of the 3'-OH of UDP-GlcNAc is to the 2-si face of FPEP, corresponding to the 2-re face of PEP. Given the previous observation that, in D2O, the addition of D+ to C-3 of PEP proceeds from the 2-si face [Kim, D. H., Lees, W. J., and Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 6380-6381], the addition across the double bond of PEP is anti. Also, because the overall stereochemical course has been shown to be either anti/syn or syn/anti [Lees, W. J., and Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 7329-7337], it now follows that the stereochemistry of elimination of H+ from C-3 and Pi from C-2 of the tetrahedral intermediate of the reaction is syn.     

Probing the active site of cytochrome P450 2B1: metabolism of 7-alkoxycoumarins by the wild type and five site-directed mutants

A series of 7-alkoxycoumarins (chain length of 1-7 carbon atoms) was utilized as active site probes of purified Escherichia coli-expressed cytochrome P450 2B1 wild type and five site-directed mutants (I114V, F206L, V363A, V363L, and G478S). The production of 7-hydroxycoumarin, the O-dealkylation product, by the wild-type enzyme exhibited a rank order of C2 > C4 > C3 > C1 > C5 > C6 = C7. The pattern observed for the P450 I114V mutant was similar to that of the wild-type enzyme, whereas with F206L and G478S mutants, the rate of O-dealkylation was low with all the compounds. In contrast, with V363A, the highest rate of product formation was observed with 7-butoxycoumarin. The V363L mutant preferentially catalyzed the O-dealkylation of 7-methoxy- and 7-ethoxycoumarin, and a further increase in the length of the alkyl chain led to a marked decrease in product formation. The stoichiometry of 7-butoxycoumarin oxidation by V363L suggested that products other than 7-hydroxycoumarin were also formed. HPLC and GC-EIMS analyses revealed that P450 2B1 V363L produced 7-(3-hydroxybutoxy)coumarin and 7-(4-hydroxybutoxy)coumarin as major oxidation products, while the V363A mutant mainly catalyzed the O-dealkylation of 7-butoxycoumarin. Docking of alkoxycoumarins into the active site of a P450 2B1 homology model confirmed the importance of the studied residues in substrate dealkylation and explained the formation of novel 7-butoxycoumarin products by the V363L mutant.     

DNA strand transfer reactions catalyzed by vaccinia topoisomerase: hydrolysis and glycerololysis of the covalent protein-DNA intermediate

Vaccinia topoisomerase forms a covalent protein-DNA intermediate at sites containing the sequence 5'-CCCTT. The T nucleotide is linked via a 3'-phosphodiester bond to Tyr-274 of the enzyme. Here, we report that the enzyme catalyzes hydrolysis of the covalent intermediate, resulting in formation of a 3'-phosphate-terminated DNA cleavage product. The hydrolysis reaction is pH-dependent (optimum pH = 9.5) and is slower, by a factor of 10(-5), than the rate of topoisomerase-catalyzed strand transfer to a 5'-OH terminated DNA acceptor strand. Mutants of vaccinia topoisomerase containing serine or threonine in lieu of the active site Tyr-274 form no detectable covalent intermediate and catalyze no detectable DNA hydrolysis. This suggests that hydrolysis occurs subsequent to formation of the covalent protein-DNA adduct and not via direct attack by water on DNA. Vaccinia topoisomerase also catalyzes glycerololysis of the covalent intermediate. The rate of glycerololysis is proportional to glycerol concentration and is optimal at pH 9.5.     

Insights into the mechanism of haemorrhage caused by snake venom metalloproteinases

Local and systemic haemorrhage are common consequences of crotaline and viperine envenoming. Several studies carried out using purified toxins have indicated that local haemorrhage can be attributed to a distinct class of venom metalloproteinases. Analyses of their cDNAs predict multi-domain enzymes, with an N-terminal metalloproteinase domain, a disintegrin-like domain and a Cys-rich C-terminus. Haemorrhagic metalloproteinases are responsible for degrading proteins of the extracellular matrix and they also have cytotoxic effects on endothelial cells. However, to date very few investigations have been carried out on the effects of venom haemorrhagic metalloproteinases on components of the haemostatic system. We describe here the effects of a high molecular weight haemorrhagic metalloproteinase, jararhagin, from the venom of a South American pit viper Bothrops jararaca, on platelet and plasma components involved in haemostasis. Jararhagin, which is not inhibited in plasma, causes the loss of the platelet collagen receptor alpha 2 beta 1 integrin (gpIa/IIa or VLA-2) and degrades the adhesive plasma protein von Willebrand factor. Alterations of these haemostatic components are known to result in bleeding. This suggests that venom haemorrhagic metalloproteinases, in addition to causing local bleeding, may also contribute to systemic haemorrhage.     

Design of fast enzymes by optimizing interaction potential in active site

The diffusional encounter between substrate and enzyme, and hence catalytic efficiency, can be enhanced by mutating charged residues on the surface of the enzyme. In this paper we present a simple method for screening such mutations. This is based on our earlier result that electrostatic enhancement of the enzyme-substrate binding rate constant can be accounted for just by the interaction potential within the active site. Assuming that catalytic and structural integrity is maintained, the catalytic efficiency can be optimized by surface charge mutations which lead to stronger interaction potential within the active site. Application of the screening method on superoxide dismutase shows that only charge mutations close to the active site will have practical effect on the catalytic efficiency. This rationalizes a large number of findings obtained in previous simulation and experimental studies.     

Insights into pigmentary phenomena provided by grafting and chimera formation in the axolotl

The expression of pigmentation patterns in axolotl pigmentary mutants was observed following three types of experimental manipulations including chimera formation, reciprocal neural crest grafts, of gonadal primordia. Three pigmentary genes were utilized including the wild type (D), white (d), and albino (a). In chimeras between white and albino embryos, melanoblasts from the white half crossed the graft interface to differentiate in albino skin. Neural crest grafts from white embryos to albinos provided melanophores of white origin that were capable of differentiation in albino skin. Grafts of gonadal primordia from albino to white embryos provided albino germ cells that formed unpigmented ovocytes together with dark ovocytes: white ovocytes from the albino grafted ovary, and dark ovocytes from the host ovary. The donor albino white ectoderm included in the graft was able to support the differentiation of melanophores, iridophores, and xanthophores that invaded the graft ectoderm from the neural crest of the white host. It was concluded that manifestation of the white or wild phenotypes may be related to the possible presence or absence of inhibiting or stimulating pigmentary factors in the skin. This possibility was discussed in the light of recent discoveries of such factors as Agouti Signaling Protein (ASP) from mammalian skin.     

Formation of a covalent Nepsilon2-guanylylhistidyl reaction intermediate by the GTP:GTP guanylyltransferase from the brine shrimp Artemia

1. Cardioplegic solutions provide the opportunity to operate on a nonbeating heart and to protect the heart against ischemic injury during cardiac surgery. The components of these solutions are constantly being modified in an effort to find the optimal solution. We studied the effects of colloidal volume replacers such as dextran, HES and gelatin as an isocolloidoosmotic addition to St. Thomas Hospital cardioplegic solution in ischemia-reperfusion injury of isolated rat hearts. 2. In the control group, after a stabilization period of 20 min, the hearts were arrested with St. Thomas Hospital cardioplegic solution for 3 min, then subjected to 30 min of global ischemia. Hearts then were reperfused for 10 min. In the experimental groups, the protocol was the same, but either HES 200/0.5 (50 g/L), modified fluid gelatin (30 g/l) or dextran 70 (25 g/L) were added to the St. Thomas Hospital solution. 3. All hearts were compared for their preischemic and postischemic contractility, heart rate, contractility rate product, coronary flow, lactate dehydrogenase, creatine phosphokinase enzyme leakage and wet/dry weight ratio. 4. All groups had similar contractility (for control, HES, gelatin and dextran groups the values at minute 10 of reperfusion were 59+/-9, 56+/-11%, 61+/-14%, 49+/-14% of initial values [P>0.05, respectively]) and enzyme leakage (lactate dehydrogenase 4.1+/-1.0, 8.1+/-1.5, 5.8+/-1.4, 3.7+/-1.2 [P>0.05] and for creatine phosphokinase 3.9+/-2.5, 6.4+/-3.7, 5.5+/-1.3, 5.5+/-0.8, P>0.05] IU xmin(-1) x g dry tissue(-1) in the reperfusion period, respectively) results as compared with the control group. 5. The addition of isocolloidoosmotic colloids to the cardioplegic solution did not appear to enhance the effectiveness of the crystalloid St. Thomas Hospital cardioplegic solution. If a colloid is to be chosen as a plasma replacer or an additive to priming solution in the preoperative period, or during open-heart surgery, it should be modified fluid gelatin-for no sign of cardiodepression was determined with the use of this agent.     

Homologies in the active site regions of lactate dehydrogenases

The persistence of viral DNA in BHK-21 cells abortively infected with human adenovirus type 12 has been investigated using reassociation kinetics. No indication of an increase in the amount of viral DNA per cell has been found. On the contrary, the amount of intracellular viral DNA sequences decreases rapidly after infection. Thus, free adenovirus type 12 DNA does not replicate in BHK-21 cells. The influence of the multiplicity of infection on the amount of persisting adenovirus type 12 DNA has also been explored. The viral DNA sequences persisting in four lines of hamster cells transformed in vitro by adenovirus type 12 at various multiplicities of infection have been quantitated and mapped by reassociation kinetics experiments using restriction endonuclease fragments of 3H-labeled adenovirus type 12 DNA. All the EcoRI restriction nuclease fragments of the adenovirus type 12 genome are represented in each of the four cell lines. Individual fragments of the viral genome are represented in multiple copies in non-equimolar amounts.     

The role of the active site cysteine in catalysis by type 1 iodothyronine deiodinase

Type 1 iodothyronine deiodinase (deiodinase 1) is a selenoenzyme that converts the prohormone T4 to the active thyroid hormone T3 by outer ring deiodination or to the inactive metabolite rT3 by inner ring deiodination. Although selenocysteine has been demonstrated to be essential for the biochemical profile of deiodinase 1, the role of a highly conserved, active site cysteine (C124 in rat deiodinase 1) has not been defined. The present studies examined the effects of a Cys124Ala mutation on rat deiodinase 1 enzymatic function and substrate affinity. At a constant 10-mM concentration of dithiothreitol (DTT), the C124A mutant demonstrated a 2-fold lower apparent maximal velocity (Vmax) and Km for rT3 (KmrT3) than the wild type for outer ring deiodination, whereas the Vmax/Km ratio was unchanged. Similarly, the apparent Vmax and KmT3 sulfate for inner ring deiodination were 2-fold lower in the C124A mutant relative to those in the wild type, with no change in the Vmax/Km ratio. The C124A mutant exhibited ping-pong kinetics in the presence of DTT, and substitution of the active site cysteine increased the KmDTT by 14-fold relative to that of the wild-type enzyme, with no significant effects on KmrT3 or Vmax. The C124A mutant was inhibited by propylthiouracil in an uncompetitive fashion and exhibited a 2-fold increase in K(i)propylthiouracil compared with that of the wild type. KmrT3 was also reduced for the C124A mutant when 5 mM reduced glutathione, a potential physiological monothiol cosubstrate, was used in outer ring deiodination assays. These results demonstrate that thiol cosubstrate interactions with C124 in type 1 deiodinase play an important role in enhancing catalytic efficiency for both outer and inner ring deiodination.     

Role of active site tyrosine residues in catalysis by human glutathione reductase

Tyr114 and Tyr197 are highly conserved residues in the active site of human glutathione reductase, Tyr114 in the glutathione disulfide (GSSG) binding site and Tyr197 in the NADPH site. Mutation of either residue has profound effects on catalysis. Y197S and Y114L have 17% and 14% the activity of the wild-type enzyme, respectively. Mutation of Tyr197, in the NADPH site, leads to a decrease in Km for GSSG, and mutation of Tyr114, in the GSSG site, leads to a decrease in Km for NADPH. This behavior is predicted for enzymes operating by a ping-pong mechanism where both half-reactions partially limit turnover. Titration of the wild-type enzyme or Y114L with NADPH proceeds in two phases, Eox to EH2 and EH2 to EH2-NADPH. In contrast, Y197S reacts monophasically, showing that excess NADPH fails to enhance the absorbance of the thiolate-FAD charge-transfer complex, the predominant EH2 form of glutathione reductase. The reductive half-reactions of the wild-type enzyme and of Y114L are similar; FAD reduction is fast (approximately 500 s-1 at 4 degreesC) and thiolate-FAD charge-transfer complex formation has a rate of 100 s-1. In Y197S, these rates are only 78 and 5 s-1, respectively. The oxidative half-reaction, the rate of reoxidation of EH2 by GSSG, of the wild-type enzyme is approximately 4-fold faster than that of Y114L. These results are consistent with Tyr197 serving as a gate in the binding of NADPH, and they indicate that Tyr114 assists the acid catalyst His467'.