Optimization of efficiency in the glyoxalase pathway

A quantitative kinetic model for the glutathione-dependent conversion of methylglyoxal to D-lactate in mammalian erythrocytes has been formulated, on the basis of the measured or calculated rate and equilibrium constants associated with (a) the hydration of methylglyoxal, (b) the specific base catalyzed formation of glutathione-(R,S)-methylglyoxal thiohemiacetals, (c) the glyoxalase I catalyzed conversion of the diastereotopic thiohemiacetals to (S)-D-lactoylglutathione, and (d) the glyoxalase II catalyzed hydrolysis of (S)-D-lactoylglutathione to form D-lactate and glutathione. The model exhibits the following properties under conditions where substrate concentrations are small in comparison to the Km values for the glyoxalase enzymes: The overall rate of conversion of methylglyoxal to D-lactate is primarily limited by the rate of formation of the diastereotopic thiohemiacetals. The hydration of methylglyoxal is kinetically unimportant, since the apparent rate constant for hydration is (approximately 500-10(3))-fold smaller than that for formation of the thiohemiacetals. The rate of conversion of methylglyoxal to (S)-D-lactoylglutathione is near optimal, on the basis that the apparent rate constant for the glyoxalase I reaction (kcatEt/Km congruent to 4-20 s-1 for pig, rat, and human erythrocytes) is roughly equal to the apparent rate constant for decomposition of the thiohemiacetals to form glutathione and methylglyoxal [k(obsd) = 11 s-1, pH 7]. The capacity of glyoxalase I to use both diastereotopic thiohemiacetals, versus only one of the diastereomers, as substrates represents a 3- to 6-fold advantage in the steady-state rate of conversion of the diastereomers to (S)-D-lactoylglutathione.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coenzyme A esters of 2-aryloxyphenoxypropionate herbicides and 2-arylpropionate antiinflammatory drugs are potent and stereoselective inhibitors of rat liver acetyl-CoA carboxylase.

The CoA esters of diclofop, haloxyfop and fluazifop are up to 425-fold more potent than the corresponding unconjugated herbicides as inhibitors of rat liver acetyl-CoA carboxylase (EC 6.4.1.2); the most potent inhibitor is (R)-fluazifopyl-CoA2 (Ki = 0.03 microM). The binding site is stereoselective for (R)-diclofop, the herbicidally active enantiomer, and for (R)-diclofopyl-CoA. The CoA esters of the antiinflammatory drugs ibuprofen and fenoprofen also strongly inhibit this carboxylase. (S)-Ibuprofenyl-CoA (Ki = 0.7 microM), the CoA ester of the enantiomer with antiinflammatory activity, is 15-fold more potent as an inhibitor than (R)-ibuprofenyl-CoA. These results suggest that some of the biological effects of these herbicides and antiinflammatory drugs in animals may be due to the inhibition of acetyl-CoA carboxylase by their acyl-CoA derivatives.     

Catalytic properties of human Lys77-plasmin. A comparative steady-state and pre-steady-state study

Values of kinetic parameters for the hydrolysis of esters and p-nitroanilides of L-lysine and L-arginine catalyzed by the Lys77 form of human plasmin (EC 3.4.21.7) have been determined between pH 5.5 and 8 (I = 0.1 M) at 21 +/- 0.5 degrees C. Over the whole pH range explored, Lys77-plasmin catalysis conforms to simple Michaelis-Menten kinetics, and steady-state and pre-steady-state data may be consistently fitted to the minimum three-step mechanism: E + S in equilibrium (k+1/k-1)E X S----(k+2)E X P + P1----(k+3)E + P2 In spite of the higher specificity of lysyl derivatives for Lys77-plasmin rather than the arginyl ones, kinetic parameters also depend on the nature of the N-alpha substituent and/or of the alcoholic or p-nitroanilidic moiety of the substrate. Among the esters and anilides considered, ZLysONp shows the most favourable kinetic parameters and may be the substrate of choice of Lys77-plasmin, in that it allows the determination of the enzyme concentration as low as 2 X 10(-9) M (about 1 X 10(-3) CU/ml), at the optimum pH value (approx. 8). Between pH 5.5 and 8, the pH profiles of kcat and kcat/Km for the Lys77-plasmin-catalyzed hydrolysis of ZLysONp and ZArgONp reflect the ionization of a single group (probably His-602 involved in the active site) with pKa values ranging between 6.4 and 6.6; at variance, values of Km are pH-independent.     

3-Hydroxy steroid dehydrogenase activities of cortisone reductase

The behaviour of various C(19) and C(18) steroids as substrates for crystalline preparations of cortisone reductase (EC 1.1.1.53) is described. 3alpha(Axial,3R)-, 3alpha(equatorial,3R)- and 3beta(axial,3S)-hydroxy steroid-NAD oxidoreductase activities are demonstrated. Four pairs of the substrates differed only in the shape of the a/b ring junction, three pairs differed only in substitution at C-10, and four pairs differed only in substitution in ring d. The shape of the substrate molecule and certain substituents (e.g. 10beta-methyl, 17beta-hydroxy, 16-oxo or 17-oxo) altered substrate behaviour, but steroids differing considerably in shape nevertheless acted as substrates, suggesting the possibility of a large or flexible binding site. K(m) values varied about 10-fold, many being approx. 140mum. V(max.) values covered a greater range (about 200-fold) and the good substrates had high V(max.) values rather than low K(m) values.     

Purification and characterization of two oxidoreductases involved in the enantioselective reduction of 3-oxo, 4-oxo and 5-oxo esters in baker's yeast

Two NADPH-dependent oxidoreductases catalyzing the enantioselective reduction of 3-oxo esters to (S)- and (R)-3-hydroxy acid esters, [hereafter called (S)- and (R)-enzymes] have been purified 121- and 332-fold, respectively, from cell extracts of Saccharomyces cerevisiae by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, Sephadex G-150 filtration, Sepharose 6B filtration and hydroxyapatite chromatography. The relative molecular mass Mr, of the (S)-enzyme was estimated to be 48,000-50,000 on Sephadex G-150 column chromatography and 48,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.9 and reduced 3-oxo esters, 4-oxo and 5-oxo acids and esters enantioselectively to (S)- hydroxy compounds in the presence of NADPH. The Km values for ethyl 3-oxobutyrate, ethyl 3-oxohexanoate, 4-oxopentanoic and 5-oxohexanoic acid were determined as 0.9 mM, 5.3 mM, 17.1 mM and 13.1 mM, respectively. The Mr of the (R)-enzyme, estimated by means of column chromatography on Sepharose 6B, was 800,000. Under dissociating conditions of SDS/polyacrylamide gel electrophoresis the enzyme resolved into subunits of Mr 200,000 and 210,000, respectively. The enzyme is optimally active at pH 6.1, catalyzing specifically the reduction of 3-oxo esters to (R)-hydroxy esters, using NADPH for coenzyme. Km values for ethyl 3-oxobutyrate and ethyl 3-oxohexanoate were determined as 17.0 mM and 2.0 mM, respectively. Investigations with purified fatty acid synthase of baker's yeast revealed that the (R)-enzyme was identical with a subunit of this multifunctional complex; intact fatty acid synthase (Mr 2.4 X 10(6)) showed no activity in catalyzing the reduction of 3-oxo esters.     

Probing the mechanism of inactivation of human pyruvate dehydrogenase by phosphorylation of three sites

Activity of the mammalian pyruvate dehydrogenase complex (PDC) is regulated by phosphorylation-dephosphorylation of three serine residues (designated site 1, Ser-264; site 2, Ser-271; site 3, Ser-203) in the alpha subunit of the pyruvate dehydrogenase (E1) component. Substitutions of the phosphorylation sites were generated by site-directed mutagenesis. Glutamate (S1E) and aspartate (S1D) substitutions at site 1 resulted in the complete loss of PDC activity; however, these mutants were variably active in the decarboxylation and 2,6-dichlorophenolindophenol assays. S1Q had only 3% of wild-type PDC activity. The apparent K(m) values for pyruvate increased for the mutants of site 1 when determined in the 2,6-dichlorophenolindophenol assay. The substitutions at sites 2 and 3 caused only moderate reductions in activity in the three assays. S3E had a 27-fold increase in the apparent K(m) for thiamine pyrophosphate and 8-fold increase in the K(i) for pyrophosphate. Site 3 was almost completely protected from phosphorylation by thiamine pyrophosphate. The results show that the size rather than negative charge of the substituted amino acid residue affects the active site of E1 and that modification of each of the three serine residues affect the active site in a site-specific manner for its ability to bind the cofactor and substrates.     

Communication between the two active sites of glutathione S-transferase A1-1, probed using wild-type-mutant heterodimers

To study the communication between the two active sites of dimeric glutathione S-transferase A1-1, we used heterodimers containing one wild-type (WT) active site and one active site with a single mutation at either Tyr9, Arg15, or Arg131. Tyr9 and Arg15 are part of the active site of the same subunit, while Arg131 contributes to the active site of the opposite subunit. The V(max) values of Tyr9 and Arg15 mutant enzymes were less than 2% that of WT, indicating their importance in catalysis. In contrast, V(max) values of Arg131 mutant enzymes were about 50-90% of that of WT enzyme while K(m)(GSH) values were approximately 3-8 times that of WT, suggesting that Arg131 plays a role in glutathione binding. The mutant enzyme (with a His(6) tag) and the WT enzyme (without a His(6) tag) were used to construct heterodimers (WT-Y9F, WT-Y9T, WT-R15Q, WT-R131M, WT-R131Q, and WT-R131E) by incubation of a mixture of wild-type and mutant enzyme at pH 7.5 in buffer containing 1,6-hexanediol, followed by dialysis against buffer lacking the organic solvent. The resultant heterodimers were separated from the wild-type and mutant homodimers using chromatography on nickel-nitrilotriacetic acid agarose. The V(max) values of all heterodimers were lower than expected for independent active sites. Our experiments demonstrate that mutation of an amino acid residue in one active site affects the activity in the other active site. Modeling studies show that key amino acid residues and water molecules connect the two active sites. This connectivity is responsible for the cross-talk between the active sites.     

Interaction between squash inhibitors and bovine trypsinogen

Squash seeds proteinase inhibitors form stoichiometric complexes with bovine trypsinogen. In terms of association constants (Ka), the interaction is weak. The inhibitors bind to the zymogen with Ka values of approx. 10(4)M-1 i.e. 2 X 10(7) times weaker than to bovine beta-trypsin. Squash inhibitor with Lys at the P1 position binds to trypsinogen with a Ka value 2.1-fold higher than the inhibitor with Arg at P1. The Ile-Val binding cleft and the Ca2+ binding site of trypsinogen are cooperatively linked to the inhibitor binding site. Although these three sites are spatially separated, either binding of calcium ion or Ile-Val dipeptide to trypsinogen increase the Ka values 3-fold and more than 100-fold, respectively. In the presence of Ile-Val trypsinogen resynthetizes extremely slowly (about 10(4) times slower than beta-trypsin) the reactive site peptide bond in squash inhibitors.     

Inhibition of glyoxalase I by the enediol mimic S-(N-hydroxy-N-methylcarbamoyl)glutathione. The possible basis of a tumor-selective anticancer strategy.

In principle, competitive inhibitors of glyoxalase I that also serve as substrates for the thioester hydrolase glyoxalase II might function as tumor-selective anti-cancer agents, given the role of these enzymes in removing cytotoxic methylglyoxal from cells and the observation that glyoxalase II activity is abnormally low in some types of cancer cells. In support of the feasibility of this anticancer strategy, an inhibitor of this type has been synthesized by a thioester-interchange reaction between glutathione and N-hydroxy-N-methylcarbamate 4-chlorophenyl ester to give S-(N-hydroxy-N-methylcarbamoyl)glutathione (1). This compound was designed to be a tight-binding inhibitor of glyoxalase I, on the basis of its stereoelectronic similarity to the enediol(ate) intermediate that forms along the reaction pathway of this enzyme. Indeed, 1 is a competitive inhibitor of yeast glyoxalase I, with an inhibition constant (Ki = 68 microM) that is approximately 30-fold lower than that reported for S-D-lactoylglutathione and approximately 7-fold lower than the Km for glutathione-methylglyoxal thiohemiacetal. In addition, 1 is a substrate for bovine liver glyoxalase II, with a Km (0.48 mM) approximately equal to that of the normal substrate S-D-lactoyglutathione and a kcat approximately 2 x 10(-5)-fold that of the normal substrate. Membrane transport studies show that 1 can be delivered into human erythrocytes (used here as a model cell) either by direct diffusion of 1 across the cell membrane or by more rapid diffusion of the glycylethyl ester of 1 across the cell membrane, followed by the catalyzed hydrolysis of the ester to give 1.     

Substrate specificity and other properties of the inducible S3 secondary alkylsulphohydrolase purified from the detergent-degrading bacterium Pseudomonas C12B.

The inducible S3 secondary alkylsulphohydrolase of the soil bacterium Pseudomonas C12B was purified to homogeneity (683-fold from cell-free extracts by a combination of column chromatography on DEAE-cellulose. Sephadex G-100 and Blue Sepharose CL-6B. The enzyme has a molecular weight in the region of 40000--46000, and is active over a broad range of pH from 5 to 9, with maximum activity at pH 8.2. The preferred substrates of the enzyme are the symmetrical secondary alkylsulphate esters such as heptan-4-yl sulphate and nonan-5-yl sulphate and the asymmetric secondary octyl and nonyl sulphate esters with the sulphate group attached to C-3 or C-4. However, for each asymmetric ester, the L-isomer is much more readily hydrolysed than the D-isomer. This specificity is interpreted in terms of a three-point attachment of the substrate to the enzyme's active site. The alkyl chains on either side of the esterified carbon atom are bound in two separate sites, one of which can only accommodate alkyl chains of limited size. The third site binds the sulphate group. Enzymic hydrolysis of this group is accompanied by complete inversion of configuration at the asymmetric carbon atom. The implied cleavage of the C--O bond of the C--O--S ester linkage was confirmed by 18O-incorporation studies.     

Purification from rat liver of an enzyme that catalyses the sulphurylation of phenols

1. An enzyme (EC 2.8.2.1) that catalyses the transfer of sulphate from adenosine 3'-phosphate 5'-sulphatophosphate to phenols was purified approx. 2000-fold from male rat livers. 2. The purified preparation did not catalyse the sulphurylation of dehydroepiandrosterone, butan-1-ol, l-tyrosine methyl ester, 1-naphthylamine or serotonin. 3. At pH8.0 and 37 degrees C the K(m) values of the enzyme for p-nitrophenol and adenosine 3'-phosphate 5'-sulphatophosphate are 51 and 14mum respectively. The K(m) value for either substrate is independent of the concentration of the other. 4. The sulphurylation of phenol is inhibited by thiol compounds and glutathione at a concentration of 3mm caused an approx. 50% decrease in enzyme activity. 5. The K(m) of the enzyme for adenosine 3'-phosphate 5'-sulphatophosphate is unaffected by the presence of added glutathione but at a concentration of 5mm-glutathione the K(m) of the enzyme for its phenolic substrate is decreased.     

Covalent modification and active site-directed inactivation of a low molecular weight phosphotyrosyl protein phosphatase.

Covalent modification experiments were conducted in order to identify active site residues of the 18-kDa cytoplasmic phosphotyrosyl protein phosphatases. The enzyme was inactivated by diethyl pyrocarbonate, phenylglyoxal, cyclohexanedione, iodoacetate, iodoacetamide, phenylarsine oxide, and certain epoxides in a manner consistent with the modification of active site residues. Phenylglyoxal and cyclohexanedione both bind to the active site in a rapid preequilibrium process and thus act as active site-directed inhibitors. The pH dependencies of the inactivation by iodoacetate and by iodoacetamide were examined in detail and compared with rate data for the alkylation of glutathione as a model compound. The enzyme inactivation data permitted the determination of pKa values of two reactive cysteines at or near the active site. Although phosphomycin is simply a competitive inhibitor of the enzyme, it was found that 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and (R)- and (S)-benzylglycidol act as irreversible covalent inactivators, consistent with the importance of a hydrophobic moiety on the substrate in controlling substrate specificity. EPNP exhibits characteristics of an active site-directed inactivator, with a preequilibrium binding constant somewhat smaller than that of phosphate ion. The pH dependencies of inactivation of EPNP and (S)-benzylglycidol are identical to that observed for iodoacetamide and similar to that for iodoacetate, suggesting that they modify similar groups. Sequencing of the tryptic digests of the EPNP-labeled enzyme indicates that Cys-62 and Cys-145 are labeled. Phenylarsine oxide acts as a very slow, tight-binding inhibitor of the enzyme. The results are interpreted in terms of an active site model that incorporates a histidine-cysteine ion pair, similar to that present in papain.     

Thiols as myeloperoxidase-oxidase substrates.

Nine low-Mr thiols were compared with regard to their ability to function as myeloperoxidase-oxidase substrates under conditions where no auto-oxidation of the thiols could be observed. The methyl and ethyl esters of cysteine were found to be about twice as active as cysteamine at pH 7.0, in terms of increased O2 consumption. Cysteine itself was poorly active, whereas glutathione, N-acetylcysteine and penicillamine were completely inactive as myeloperoxidase-oxidase substrates under these conditions. The structure-activity relationships indicated that both a free thiol and free amino group were required for peroxidase-oxidase activity, and also that a free carboxy group abolished activity. In analogy with cysteamine, the activities of both cysteine esters were inhibited by superoxide dismutase (less than 5 micrograms/ml) and by catalase and not by the hydroxyl-radical scavenger mannitol. In contrast with cysteamine, the activities of both cysteine esters were stimulated more than 2-fold by high concentrations (greater than 5 micrograms/ml) of superoxide dismutase. The activities of both cysteine esters exhibited broad pH optima at pH 7. A mechanism for the myeloperoxidase-oxidase oxidation of the cysteine esters is proposed, which is partly different from that previously proposed for cysteamine.     

Urokinase-catalysed plasminogen activation. Effects of ligands binding to the AH-site of plasminogen

The kinetics of activation of Lys-plasminogen (Lys-77-Asn-790) and miniplasminogen (Val-442-Asn-790) catalysed by low-molecular-weight urokinase (LMW-urokinase) was investigated in the presence and absence of ligands that bind to the AH-site of the plasminogens. 6-Aminohexanoic acid and alpha-N-acetyl-L-lysine methyl ester (AcLysMe) were used. Saturation of the AH-sites of the plasminogens result in similar, but rather small positive effects on the kinetics of activation of the two plasminogens. Michaelis constants decrease approx. 2-fold and second-order rate constants (kc/Km)Pg increase approx. 1.2-fold. Michaelis constants (KPg values) were obtained using a new approach; the values were determined from the competing effects of the plasminogens on urokinase-catalysed hydrolysis of a synthetic substrate. In the pH range 7.4-8.0, only minor alterations of the values of the kinetic parameters are observed. At 25 degrees C, values of (kc/Km)Pg are approx. 3-fold less than the value at 37 degrees C, whereas KPg is not changed. We conclude that kc/Km values are approx. 10(5) M-1.s-1 and that KPg values are approx. 40 microM of urokinase-catalysed conversions of Lys- and miniplasminogen to their respective plasmins.     

The active-site residue tyr-175 in human glyoxalase II contributes to binding of glutathione derivatives

Tyrosine-175 located in the active site of human glyoxalase II was replaced by phenylalanine in order to study the contribution of this residue to catalysis. The mutation had a marginal effect on the k(cat) value determined using S-D-lactoylglutathione as substrate. However, the Y175F mutant had an 8-fold higher K(m) value than the wild-type enzyme. The competitive inhibitor S-(N-hydroxy-N-bromophenylcarbamoyl)glutathione had a 30-fold higher K(i) value towards the mutant, than that of the wild-type. Pre-equilibrium fluorescence studies with the inhibitor showed that this was due to a significantly increased off-rate for the mutant enzyme. The phenolic hydroxyl group of tyrosine-175 is within hydrogen bonding distance of the amide nitrogen of the glycine in the glutathione moiety and the present study shows that this interaction makes a significant contribution to the binding of the active-site ligand.     

Role of the glutamyl alpha-carboxylate of the substrate glutathione in the catalytic mechanism of human glutathione transferase A1-1.

The Glu alpha-carboxylate of glutathione contributes to the catalytic function of the glutathione transferases. The catalytic efficiency of human glutathione transferase A1-1 (GST A1-1) in the conjugation reaction with 1-chloro-2,4-dinitrobenzene is reduced 15 000-fold if the decarboxylated analogue of glutathione, dGSH (GABA-Cys-Gly), is used as an alternative thiol substrate. The decrease is partially due to an inability of the enzyme to promote ionization of dGSH. The pK(a) value of the thiol group of the natural substrate glutathione decreases from 9.2 to 6.7 upon binding to GST A1-1. However, the lack of the Glu alpha-carboxylate in dGSH raised the pK(a) value of the thiol in the enzymatic reaction to that of the nonenzymatic reaction. Furthermore, K(M)(dGSH) was 100-fold higher than K(M)(GSH). The active-site residue Thr68 forms a hydrogen bond to the Glu alpha-carboxylate of glutathione. Introduction of a carboxylate into GST A1-1 by a T68E mutation increased the catalytic efficiency with dGSH 10-fold and reduced the pK(a) value of the active site bound dGSH by approximately 1 pH unit. The altered pK(a) value is consistent with a catalytic mechanism where the carboxylate contributes to ionization of the glutathione thiol group. With Delta(5)-androstene-3,17-dione as substrate the efficiency of the enzyme is decreased 24 000-fold while with 4-nitrocinnamaldehyde (NCA) the decrease is less than 150-fold. In the latter reaction NCA accepts a proton and, unlike the other reactions studied, may not be dependent on the Glu alpha-carboxylate for deprotonation of the thiol group. An additional function of the Glu alpha-carboxylate may be productive orientation of glutathione within the active site.     

Identification of fructose 6-phosphate- and fructose 1-phosphate-binding residues in the regulatory protein of glucokinase.

Glucokinase is inhibited in the liver by a regulatory protein (GKRP) whose effects are increased by Fru-6-P and suppressed by Fru-1-P. To identify the binding site of these phosphate esters, we took advantage of the homology of GKRP to the isomerase domain of GlmS (glucosamine-6-phosphate synthase) and created 12 different mutants of rat GKRP. Mutations of three residues predicted to bind to Fru-6-P resulted in proteins that were approximately 5-fold (S110A) and 50-fold (S179A and K514A) less potent as inhibitors of glucokinase and had an at least 100-fold reduced affinity for the effectors. Mutation of another residue of the putative binding site (T109A) resulted in a 10-fold decrease in the inhibitory power and an inversion of the effect of sorbitol-6-P, a Fru-6-P analog. The replacement of Gly(107), a residue close to the binding site, by cysteine (as in GlmS and Xenopus GKRP) resulted in a protein that had 20 times more affinity for Fru-6-P and 30 times less affinity for Fru-1-P. These results are consistent with GKRP having one single binding site for phosphate esters. They also show that a missense mutation of GKRP can lead to a gain of function.     

Investigation of invariant serine/threonine residues in mevalonate kinase. Tests of the functional significance of a proposed substrate binding motif and a site implicated in human inherited disease

Mevalonate kinase serine/threonine residues have been implicated in substrate binding and inherited metabolic disease. Alignment of >20 mevalonate kinase sequences indicates that Ser-145, Ser-146, Ser-201, and Thr-243 are the only invariant residues with alcohol side chains. These residues have been individually mutated to alanine. Structural integrity of the mutants has been demonstrated by binding studies using fluorescent and spin-labeled ATP analogs. Kinetic characterization of the mutants indicates only modest changes in K(m)((ATP)). K(m) for mevalonate increases by approximately 20-fold for S146A, approximately 40-fold for T243A, and 100-fold for S201A. V(max) changes for S145A, S201A, and T243A are < or =3-fold. Thus, the 65-fold activity decrease associated with the inherited human T243I mutation seems attributable to the nonconservative substitution rather than any critical catalytic function. V(max) for S146A is diminished by 4000-fold. In terms of V/K(MVA), this substitution produces a 10(5)-fold effect, suggesting an active site location and catalytic role for Ser-146. The large k(cat) effect suggests that Ser-146 productively orients ATP during catalysis. K(D(Mg-ATP)) increases by almost 40-fold for S146A, indicating a specific role for Ser-146 in liganding Mg(2+)-ATP. Instead of mapping within a proposed C-terminal ATP binding motif, Ser-146 is situated in a centrally located motif, which characterizes the galactokinase/homoserine kinase/ mevalonate kinase/phosphomevalonate kinase protein family. These observations represent the first functional demonstration that this region is part of the active site in these related phosphotransferases.     

A conserved serine juxtaposed to the pseudosubstrate site of type I cGMP-dependent protein kinase contributes strongly to autoinhibition and lower cGMP affinity

Serines 64 and 79 are homologous residues that are juxtaposed to the autoinhibitory pseudosubstrate site in cGMP-dependent protein kinase type Ialpha and type Ibeta (PKG-Ialpha and PKG-Ibeta), respectively. Autophosphorylation of this residue is associated with activation of type I PKGs. To determine the role of this conserved serine, point mutations have been made in PKG-Ialpha (S64A, S64T, S64D, and S64N) and PKG-Ibeta (S79A). In wild-type PKG-Ialpha, basal kinase activity ratio (-cGMP/+cGMP) is 0.11, autophosphorylation increases this ratio 3-fold, and the K(a) and K(D) values for cGMP are 127 and 36 nm, respectively. S64A PKG-Ialpha basal kinase activity ratio increases 2-fold, cGMP binding affinity increases approximately 10-fold in both K(a) and K(D), and activation by autophosphorylation is slight. S64D and S64N mutants are nearly constitutively active in the absence of cGMP, cGMP binding affinity in each increases 18-fold, and autophosphorylation does not affect the kinase activity of these mutants. Mutation of the homologous site in PKG-Ibeta (S79A) increases the basal kinase activity ratio 2-fold and cGMP binding affinity 5-fold over that of wild-type PKG-Ibeta. The combined results demonstrate that a conserved serine juxtaposed to the pseudosubstrate site in type I PKGs contributes importantly to enzyme function by increasing autoinhibition and decreasing cGMP binding affinity.     

Inhibition of malic enzyme by S-oxalylglutathione, a probable in vivo effector

Various oxalyl thiol esters (RSCOCOO-), especially S-oxalylglutathione (GS-Ox), were found to be very effective inhibitors of chicken liver malic enzyme. When the conditions are similar to those encountered physiologically [high reduced nicotinamide adenine dinucleotide phosphate (NADPH) concentrations], inhibition is detectable with less than 1 microM concentrations of GS-Ox. The amount of inhibition is not reversed by excess glutathione, thus indicating that it is not due to oxalyl transfer to some enzymic thiol group with release of glutathione. Detailed kinetic studies show that the inhibition by GS-Ox can be treated as a simple reversible binding to the enzyme; the double reciprocal plot patterns indicate that the inhibition is linear noncompetitive (mixed type), vs. both L-malate in the oxidative decarboxylation reaction and pyruvate in the reverse reaction. At pH 7.4 and 25 degrees C in the presence of 100-200 microM NADPH, the Kis and Kii values for GS-Ox are 0.7 and 5 microM, respectively, and are the same for reactions run in either direction. The high specificity for GS-Ox is indicated by the observation that, under similar conditions, the Kis values for S-oxalyl coenzyme A and S-oxalyl-N-acetylcysteamine are 40 and 150 microM, respectively. Such high specificity indicates that the enzyme has evolved a specific binding site for the glutathione part of GS-Ox. The current results, when considered in conjunction with recent evidence that oxalyl thiol esters are present in animal tissues at concentrations up to 50 microM, imply that GS-Ox is an important in vivo regulator of malic enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)