Studies on regulatory functions of malic enzymes. VII. Structural and functional characteristics of sulfhydryl groups in NADP-linked malic enzyme from Escherichia coli W.

NADP-linked malic enzyme from Escherichia coli W contains 7 cysteinyl residues per enzyme subunit. The reactivity of sulfhydryl (SH) groups of the enzyme was examined using several SH reagents, including 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM). 1. Two SH groups in the native enzyme subunit reacted with DTNB (or NEM) with different reaction rates, accompanied by a complete loss of the enzyme activity. The second-order modification rate constant of the "fast SH group" with DTNB coincided with the second-order inactivation rate constant of the enzyme by the reagent, suggesting that modification of the "fast SH group" is responsible for the inactivation. When the enzyme was denatured in 4 M guanidine HCl, all the SH groups reacted with the two reagents. 2. Althoug the inactivation rate constant was increased by the addition of Mg2+, an essential cofactor in the enzyme reaction, the modification rate constant of the "fast SH group" was unaffected. The relationship between the number of SH groups modified with DTNB or NEM and the residual enzyme activity in the absence of Mg2+ was linear, whereas that in the presence of Mg2+ was concave-upwards. These results suggest that the Mg2+-dependent increase in the inactivation rate constant is not the result of an increase in the rate constant of the "fast FH group" modification. 3. The absorption spectrum of the enzyme in the ultraviolet region was changed by addition of Mg2+. The dissociation constant of the Mg2+-enzyme complex obtained from the Mg2+- dependent increment of the difference absorption coincided with that obtained from the Mg2+- dependent enhancement of NEM inactivation. 4. Both the inactivation rate constant and the modification rate constant of the "fast SH group" were decreased by the addition of NADP+. The protective effect of NADP+ was increased by the addition of Mg2+. Based on the above results, the effects of Mg2+ on the SH-group modification are discussed from the viewpoint of conformational alteration of the enzyme.     

Functional cysteinyl residues in human placental aldose reductase

Incubation of human placental aldose reductase (EC 1.1.1.21) with the sulfhydryl oxidizing reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM) results in a biexponential loss of catalytic activity. Inactivation by DTNB or NEM is prevented by saturating concentrations of NADPH. ATP-ribose offers partial protection against inactivation by DTNB, whereas NADP, nicotinamide mononucleotide (NMN), and the substrates glyceraldehyde and glucose offer little or no protection. The inactivation by DTNB was reversed by dithiothreitol and partially by 2-mercaptoethanol but not by KCN. When the release of 2-nitro-5-mercaptobenzoic acid was measured, 3 mol of sulfhydryl residues was found to be modified per mole of the enzyme by DTNB. Correlation of the fractional activity remaining with the extent of modification by the statistical method of C.-L. Tsou (1962, Sci. Sin. 11, 1535-1558) indicates that of the three reactive residues, one reacts at a faster rate than the other two, and that two residues are essential for the catalytic activity of the enzyme. Labeling of the total sulfhydryl by [14C]NEM and quantification of DTNB-reactive residues in the enzyme denatured by 6 M urea indicates that a total of seven sulfhydryl residues are present in the protein. The modification of the enzyme did not affect Km glyceraldehyde, but the modified enzyme had a lower Km NADPH. Kinetic analysis of the data suggests that a biexponential nature of inactivation could be due to the formation of a dissociable E:DTNB complex and the presence of a partially active enzyme species.     

Reactivities of sulfhydryl groups in native and metal-free aminoacylase I

Aminoacylase I from porcine kidney (EC 3.5.1.14) contains seven cysteine residues per subunit. Three sulfhydryl groups are accessible to modification by 4-hydroxymercuribenzoate (p-MB). The kinetics of the reaction suggest that only one of these groups affects acylase activity when modified by p-MB. Its reaction rate increases 2-3-fold when the essential metal ion of aminoacylase is removed. Modification of metal-free apoenzyme by N-ethylmaleimide (NEM) abolishes its activity without impairing Zn2+ binding. This indicates that the sulfhydryl group reacting with NEM is not directly coordinated to the metal. DTNB (5,5'-Dithio-bis(2-nitrobenzoate), Ellman's reagent) also modifies three sulfhydryl groups per subunit. In this case, the reactivities of native aminoacylase and apoenzyme are not significantly different. N-Hydroxy-2-aminobutyrate, a strong aminoacylase inhibitor, substantially increases the reactivity of the slowest reacting sulfhydryl in both native enzyme and metal-free aminoacylase. It appears that binding of the inhibitor or removal of the metal ion induces conformational changes of the amino-acylase active site that render a buried sulfhydryl group more accessible to modification.     

Chemical modification of acyl-CoA:cholesterol O-acyltransferase. 2. Identification of a coenzyme A regulatory site by p-mercuribenzoate modification

Acyl-CoA:cholesterol O-acyltransferase (EC 2.3.1.26, ACAT) is the major intracellular cholesterol-esterifying activity in vascular tissue and is potentially a key regulator of intracellular cholesterol homeostasis during atherogenesis. We have previously reported inhibition of microsomal ACAT by histidine and sulfhydryl-selective chemical modification reagents and present here a more detailed analysis of the effect of sulfhydryl modification on ACAT activity. This analysis indicated two effects of sulfhydryl modification on ACAT activity. Modification of aortic microsomes with relatively low concentrations of p-mercuribenzoate (PMB) (100-200 microM) identified an inhibitory coenzyme A binding site on ACAT which contains a modifiable sulfhydryl group. This site binds CoA tightly (Ki = 20 microM), and PMB modification prevented subsequent ACAT inhibition by CoA without itself inhibiting enzyme activity. At higher concentrations (1-2 mM), PMB inhibited ACAT activity, indicating the presence of a modifiable sulfhydryl group necessary for cholesterol esterification by ACAT. Modification of both sites by PMB was reversible by thiols, and protection against modification was afforded in both cases by oleoyl-CoA, indicating that these sites may also bind oleoyl-CoA. Thus, at least two sulfhydryl groups influence ACAT activity: one is necessary for cholesterol esterification by ACAT, and one is at or near an inhibitory CoA binding site, which may be occupied at intracellular concentrations of CoA.     

Studies on phosphoglyceromutase from chicken breast muscle: number and reactivity of sulfhydryl groups.

Phosphoglyceromutase (PGM) from chicken breast muscle was titrated with p-mercuribenzoate (PMB), 5,5'-dithiobisnitrobenzoate (Nbs2), N-ethylmaleimide (NEM), iodoacetate and iodoacetamide. The effect of all of the sulfhydryl reagents, with the exception of NEM was to cause a loss in enzymatic activity. Addition of KCN following reaction with Nbs2 resulted in the recovery of a small amount of enzymatic activity. In the absence of substrate (3-phosphoglyceric acid) or cofactor (2,3-diphosphoglyceric acid) and in the presence or absence of 6 M guanidine hydrochloride, six sulfhydryl groups per mole of enzyme were titrated with PMB.     

Sulfhydryl groups of an extramitochondrial acetyl-CoA hydrolase from rat liver

An extramitochondrial acetyl-CoA hydrolase (EC 3.1.2.1) purified from rat liver was inactivated by heavy metal cations (Hg2+, Cu2+, Cd2+ and Zn2+), which are known to be highly reactive with sulfhydryl groups. Their order of potency for enzyme inactivation was Hg2+ greater than Cu2+ greater than Cd2+ greater than Zn2+. This enzyme was also inactivated by various sulfhydryl-blocking reagents such as p-hydroxymercuribenzoate (PHMB), N-ethylmaleimide (NEM), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and iodoacetate (IAA). DL-Dithiothreitol (DTT) reversed the inactivation of this enzyme by DTNB markedly, and that by PHMB slightly, but did not reverse the inactivations by NEM, DTNB and IAA. Benzoyl-CoA (a substrate-like competitive inhibitor) and ATP (an activator) greatly protected acetyl-CoA hydrolase from inactivation by PHMB, NEM, DTNB and IAA. These results suggest that the essential sulfhydryl groups are on or near the substrate binding site and nucleotide binding site. The enzyme contained about four sulfhydryl groups per mol of monomer, as estimated with DTNB. When the enzyme was denatured by 4 M guanidine-HCl, about seven sulfhydryl groups per mol of monomer reacted with DTNB. Two of the four sulfhydryl groups of the subunit of the native enzyme reacted with DTNB first without any significant inactivation of the enzyme, but its subsequent reaction with the other two sulfhydryl groups seemed to be involved in the inactivation process.     

A comparative study of sulfhydryl groups required for the catalytic activity of gramicidin S synthetase and isoleucyl tRNA synthetase

The sulfhydryl groups required for the catalytic activity of gramicidin S synthetase of Bacillus brevis and Escherichia coli isoleucyl tRNA synthetase were compared. In gramicidin S synthetase 2(GS 2), about four sulfhydryl groups react rapidly with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or N-ethylmaleimide (NEM), and are essential for gramicidin S formation in the presence of gramicidin S synthetase 1 (GS 1). These sulfhydryl groups are protected against DTNB and NEM reactions by the preincubation of GS 2 with amino acid substrates in the presence of ATP and MgCl2, like the sulfhydryl groups that react rapidly with DTNB or NEM and are required for the catalytic activity of GS 1 and isoleucyl tRNA synthetase. In GS 2, GS 1, and isoleucyl tRNA synthetase, the sulfhydryl group that reacts rapidly with NEM and is required for the catalytic activity is involved in the amino acid binding as a thioester. In isoleucyl tRNA synthetase, it is suggested that isoleucine may be transferred from the isoleucine thioester enzyme complex to tRNA by a mechanism similar to that proposed for gramicidin S synthetase.     

Conformational features of bovine heart mitochondrial transhydrogenase

Both purified and functionally reconstituted bovine heart mitochondrial transhydrogenase were treated with various sulfhydryl modification reagents in the presence of substrates. In all cases, NAD+ and NADH had no effect on the rate of inactivation. NADP+ protected transhydrogenase from inactivation by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in both systems, while NADPH slightly protected the reconstituted enzyme but stimulated inactivation in the purified enzyme. The rate of N-ethylmaleimide (NEM) inactivation was enhanced by NADPH in both systems. The copper-(o-phenanthroline)2 complex [Cu(OP)2] inhibited the purified enzyme, and this inhibition was substantially prevented by NADP+. Transhydrogenase was shown to undergo conformational changes upon binding of NADP+ or NADPH. Sulfhydryl quantitation with DTNB indicated the presence of two sulfhydryl groups exposed to the external medium in the native conformation of the soluble purified enzyme or after reconstitution into phosphatidylcholine liposomes. In the presence of NADP+, one sulfhydryl group was quantitated in the nondenatured soluble enzyme, while none was found in the reconstituted enzyme, suggesting that the reactive sulfhydryl groups were less accessible in the NADP+-enzyme complex. In the presence of NADPH, however, four sulfhydryl groups were found to be exposed to DTNB in both the soluble and reconstituted enzymes. NEM selectively reacted with only one sulfhydryl group of the purified enzyme in the absence of substrates, but the presence of NADPH stimulated the NEM-dependent inactivation of the enzyme and resulted in the modification of three additional sulfhydryl groups. The sulfhydryl group not modified by NEM in the absence of substrates is not sterically hindered in the native enzyme as it can still be quantitated by DTNB or modified by iodoacetamide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of Escherichia coli glycerol kinase by 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide: evidence for nucleotide regulatory binding sites

Glycerol kinase (EC 2.7.1.30, ATP:glycerol 3-phosphotransferase) from Escherichia coli is inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and by N-ethylmaleimide (NEM) in 0.1 M triethanolamine at pH 7 and 25 degrees C. The inactivation by DTNB is reversed by dithiothreitol. In the cases of both reagents, the kinetics of activity loss are pseudo first order. The dependencies of the rate constants on reagent concentration show that while the inactivation by NEM obeys second-order kinetics (k2app = 0.3 M-1 s-1), DTNB binds to the enzyme prior to the inactivation reaction; i.e., the pseudo-first-order rate constant shows a hyperbolic dependence on DTNB concentration. Complete inactivation by each reagent apparently involves the modification of two sulfhydryl groups per enzyme subunit. However, analysis of the kinetics of DTNB modification, as measured by the release of 2-nitro-5-thiobenzoate, shows that the inactivation is due to the modification of one sulfhydryl group per subunit, while two other groups are modified 6 and 15 times more slowly. The enzyme is protected from inactivation by the ligands glycerol, propane-1,2-diol, ATP, ADP, AMP, and cAMP but not by Mg2+, fructose 1,6-bisphosphate, or propane-1,3-diol. The protection afforded by ATP or AMP is not dependent on Mg2+. The kinetics of DTNB modification are different in the presence of glycerol or ATP, despite the observation that the degree of protection afforded by both of these ligands is the same.(ABSTRACT TRUNCATED AT 250 WORDS)     

Topography and chemical reactivity of the active-inactive transition-sensitive SH-group in the mitochondrial NADH:ubiquinone oxidoreductase (Complex I)

The spatial arrangement and chemical reactivity of the activation-dependent thiol in the mitochondrial Complex I was studied using the membrane penetrating N-ethylmaleimide (NEM) and non-penetrating anionic 5,5'-dithiobis-(2-nitrobenzoate) (DTNB) as the specific inhibitors of the enzyme in mitochondria and inside-out submitochondrial particles (SMP). Both NEM and DTNB rapidly inhibited the de-activated Complex I in SMP. In mitochondria NEM caused rapid inhibition of Complex I, whereas the enzyme activity was insensitive to DTNB. In the presence of the channel-forming antibiotic alamethicin, mitochondrial Complex I became sensitive to DTNB. Neither active nor de-activated Complex I in SMP was inhibited by oxidized glutathione (10 mM, pH 8.0, 75 min). The data suggest that the active/de-active transition sulfhydryl group of Complex I which is sensitive to inhibition by NEM is located at the inner membrane-matrix interface. These data include the sidedness dependency of inhibition, effect of pH, ionic strength, and membrane bilayer modification on enzyme reactivity towards DTNB and its neutral analogue.     

Topography and chemical reactivity of the active–inactive transition-sensitive SH-group in the mitochondrial NADH:ubiquinone oxidoreductase (Complex I)

The spatial arrangement and chemical reactivity of the activation-dependent thiol in the mitochondrial Complex I was studied using the membrane penetrating N-ethylmaleimide (NEM) and non-penetrating anionic 5,5′-dithiobis-(2-nitrobenzoate) (DTNB) as the specific inhibitors of the enzyme in mitochondria and inside-out submitochondrial particles (SMP). Both NEM and DTNB rapidly inhibited the de-activated Complex I in SMP. In mitochondria NEM caused rapid inhibition of Complex I, whereas the enzyme activity was insensitive to DTNB. In the presence of the channel-forming antibiotic alamethicin, mitochondrial Complex I became sensitive to DTNB. Neither active nor de-activated Complex I in SMP was inhibited by oxidized glutathione (10 mM, pH 8.0, 75 min). The data suggest that the active/de-active transition sulfhydryl group of Complex I which is sensitive to inhibition by NEM is located at the inner membrane–matrix interface. These data include the sidedness dependency of inhibition, effect of pH, ionic strength, and membrane bilayer modification on enzyme reactivity towards DTNB and its neutral analogue.     

Modification of the erythrocyte membrane by sulfhydryl group reagents

Summary In this study, the consequences of modification of human erythrocyte membrane sulfhydryl groups by N-ethyl maleimide (NEM), 5,5dithiobis-(2-nitrobenzoic acid) (DTNB) andp-hydroxymercuriphenyl sulfonate (PHMPS) were investigated. These reagents differ in chemical reactivity, membrane penetrability and charge characteristics.Results of sulfhydryl modification were analyzed in terms of inhibitory effects on activities of five membrane enzymes; Mg⁺⁺- and Na⁺, K⁺-ATPase, K⁺-dependent and independentp-nitrophenyl phosphatase (NPPase) and DPNase. Structural considerations involved in the sulfhydryl-mediated inhibition were evaluated by studying the changes in susceptibility to sulfhydryl alteration produced by shearing membranes into microvesicles and by the addition of the membrane modifiers, Mg⁺⁺ and ATP.Conclusions from the data suggest that the effects of NEM appeared to result from modification of a single class of sulfhydryls; DTNB interacted with two different sulfhydryl classes. Increasing concentrations of PHMPS resulted in the sequential modification of many types of sulfhydryls, presumably as a result of increasing membrane structural disruption. DTNB and PHMPS caused solubilization of about 15% of membrane protein at concentrations giving maximal enzyme inhibition.In contrast to the usually observed parallels between Na⁺, K⁺-ATPase and K⁺-dependent NPPase, activities of Mg⁺⁺-ATPase, Na⁺, K⁺-ATPase and K⁺-dependent NPPase varied independently as a result of sulfhydryl modification. We suggest complex structural and functional relationships exist among these components of the membrane ATP-hydrolyzing system.Our studies indicate that the effects of sulfhydryl group reagents on these membrane systems should not be ascribed to sulfhydryl modificationper se, but rather to the resulting structural perturbations. These effects depend upon the structural characteristics of the particular membrane preparation studied and on the chemical characteristics of the sulfhydryl group reagent used.     

Reactions of the sulfhydryl groups of alanyl-tRNA synthetase

The six sulfhydryl groups in each subunit of the alanyl-tRNA synthetase of Escherichia coli react with sulfhydryl reagents with at least four different rates. One reacts very rapidly with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), and a second reacts somewhat less rapidly with this reagent. These two groups are required for transfer activity, which is lost in proportion to the extent of derivatization. Two other groups react more slowly, with a consequent loss of exchange activity. The remaining two sulfhydryl groups do not react with DTNB until the protein is denatured. The inactivations are reversed by dithiothreitol. Two sulfhydryl groups react with N-ethylmaleimide (NEM) and with a spin-label derivative of NEM. These reactions resemble the modification of two sulfhydryl groups with DTNB, in that they also inactivate the transfer reaction but not the ATP:PP_i exchange. The two spin labels are incorporated at similar rates but are in very different environments, one highly exposed and one highly immobilized. These groups do not interact with Mn²⁺, which is bound to the enzyme in the absence of ATP.     

Role of the reactive cysteine residue in restriction endonuclease Cfr9I.

Chemical modification studies were performed to elucidate the role of Cys-residues in the catalysis/binding of restriction endonuclease Cfr9I. Incubation of restriction endonuclease Cfr9I with N-ethylmaleimide (NEM), iodoacetate, 5,5'-dithiobis (2-nitrobenzoic acid) at pH 7.5 led to a complete loss of the catalytic activity. However, no enzyme inactivation was detectable after modification of the enzyme with iodoacetamide and methyl methanethiosulfonate. Complete protection of the enzyme against inactivation by NEM was observed in the presence of substrate implying that Cys-residues may be located at or in the vicinity of the active site of enzyme. Direct substrate-binding studies of native and modified restriction endonuclease Cfr9I using a gel-mobility shift assay indicated that the modification of the enzyme by NEM was hindered by substrate binding. A single Cys-residue was modified during the titration of the enzyme with DTNB with concomitant loss of the catalytic activity. The pH-dependence of inactivation of Cfr9I by NEM revealed the modification of the residue with the pKa value of 8.9 +/- 0.2. The dependence of the reaction rate of substrate hydrolysis by Cfr9I versus pH revealed two essential residues with pKa values of 6.3 +/- 0.15 and 8.7 +/- 0.15, respectively. The evidence presented suggests that the restriction endonuclease Cfr9I contains a reactive sulfhydryl residue which is non-essential for catalysis, but is located at or near the substrate binding site.     

Modification of the adipocyte lipid binding protein by sulfhydryl reagents and analysis of the fatty acid binding domain

The adipocyte lipid binding protein (ALBP) is a member of a multigene family of low molecular weight proteins which stoichiometrically and saturably bind hydrophobic ligands and presumably facilitate intracellular lipid metabolism. To probe the structure-function relationship of the binding domain of ALBP, chemical modification has been employed. Modification of the two cysteinyl residues of ALBP (Cys1 and Cys117) with a variety of sulfhydryl reagents decreased the apparent affinity for oleic acid in the following order of effectiveness: methyl methanethiosulfonate much much less than p-(chloromercuri)benzenesulfonic acid less than N-ethylmaleimide (NEM) = 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Thiol titration of ALBP with DTNB in the presence of bound oleate resulted in the modification of a single cysteinyl residue. The oleate-protected cysteine was identified as Cys117 by modification with a combination of reversible (DTNB) and irreversible (NEM) sulfhydryl reagents in the presence or absence of saturating oleic acid. Cys117-NEM ALBP exhibited a large decrease in binding affinity while Cys1-NEM ALBP exhibited normal binding properties. Neither the modification of ALBP with NEM nor the addition of oleic acid had a significant effect on protein structure, as judged by circular dichroic analysis. These results suggest that Cys117 of ALBP resides in the ligand binding domain and that site-specific modification can be utilized to assess the conformational flexibility of the binding cavity.     

Studies on aspartase. II. Role of sulfhydryl groups in aspartase from Escherichia coli.

Aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) of Escherichia coli W contains 38 half-cystine residues per tetrameric enzyme molecule. Two sulfhydryl groups were modified with N-ethylmaleimide or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) per subunit, while 8.3 sulfhydryl groups were titrated with p-mercuribenzoic acid. In the presence of 4 M guanidine - HCl, 8.6 sulfhydryl groups reacted with DTNB per subunit. Aspartase was inactivated by various sulfhydryl reagents following pseudo-first-order kinetics. Upon modification of one sulfhydryl group per subunit with N-Ethylmaleimide, 85% of the original activity was lost; a complete inactivation was attained concomitant with the modification of two sulfhydryl groups. These results indicate that one or two sulfhydryl groups are essential for enzyme activity. L-Aspartate and DL-erythro-beta-hydroxyaspartate markedly protected the enzyme against N-ethylmaleimide-inactivation. Only the compounds having an amino group at the alpha-position exhibited protection, indicating that the amino group of the substrate contributes to the protection of sulfhydryl groups of the enzyme. Examination of enzymatic properties after N-ethylmaleimide modification revealed that 5-fold increase in the Km value for L-aspartate and a shift of the optimum pH for the activity towards acidic pH were brought about by the modification, while neither dissociation into subunits nor aggregation occurred. These results indicate that the influence of the sulfhydryl group modification is restricted to the active site or its vicinity of the enzyme.     

The reactivity of sulfhydryl groups of yeast DNA dependent RNA polymerase I

The number of reactive cysteine residues of yeast RNA polymerase I was determined and their function was studied using parachloromercury benzoate (pCMB), dithiobisnitrobenzoate (DTNB) and N-ethyl-maleimide (NEM) as modifying agents. By treatment with DTNB about 45 sulfhydryl groups react in the presence of 8M urea. Under non-denaturing conditions only 20 sulfhydryl groups are reactive with pCMB and DTNB. Both reagents completely inactivate the enzyme and this effect can be reversed by reducing agents. The sedimentation coefficient and the subunit composition are not affected when the enzyme is inactivated. Two of the most reactive sulfhydryl groups are necessary for activity. The modification of these groups is partially protected by substrates and DNA, suggesting that they are involved either in catalysis or in the maintenance of the conformation of the active site. Experiments with 14C-NEM indicate that the most reactive groups are located in subunits of 185,000, 137,000 and 41,000 daltons.     

Studies on regulatory functions of malic enzymes. VI. Purification and molecular properties of NADP-linked malic enzyme from Escherichia coli W.

NADP-linked malic enzyme [EC 1.1.1.40] was highly purified from Escherichia coli W cells. The purified enzyme was homogeneous as judged by ultracentrifugation and gel electrophoresis. The apparent molecular weights obtained by sedimentation equilibrium analysis, from diffusion and sedimentation constants, and by disc electrophoresis at various gel concentrations were 471,000, 438,000, and 495,000, respectively. The subunit molecular weights obtained by sedimentation equilibrium analysis in the presence of 6 M guanidine hydrochloride and gel electrophoresis in the presence of sodium dodecyl sulfate were 76,000 and 82,000, respectively. The sedimentation coefficient (S(0)20, W) was 13.8S, and the molecular activity was 44,700 min-1 at 30 degrees C. The amino acid composition of the enzyme was determined, and the results were compared with those of NAD-linked malic enzyme from the same organism and those of pigeon liver NADP-linked malic enzyme. The partial specific volume was calculated to be 0.738 ml/g. The Km value for L-malate was 2.3 mM at pH 7.4. Malonate, tartronate, glutarate, and DL-tartrate competitively inhibited the activity. The saturation profile for L-malate exhibited a marked cooperativity in the presence of both chloride ions and acetyl-CoA. However, acetyl-CoA alone did not show cooperativity or produce inhibition in the absence of chloride ions. Vmax and Km were determined as a function of pH. The optimum pH for the reaction was 7.8. Inspection of the Dixon plots suggested that three ionizable groups of the enzyme are essential for the enzyme activity. In addition to the oxidative decarboxylase activity, the enzyme preparation exhibited divalent metal ion-dependent oxaloacetate decarboxylase and alpha-keto acid reductase activities. Based on the above results, the molecular properties of the enzymatic reaction are discussed.     

Deoxycytidylate hydroxymethylase: purification, properties, and the role of a thiol group in catalysis

Deoxycytidylate (dCMP) hydroxymethylase from Escherichia coli infected with a T-4 bacteriophage amber mutant has been purified to homogeneity. It is a dimer with a subunit molecular weight of 28,000. Chemical modification of the homogeneous enzyme with N-ethylmaleimide (NEM) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) leads to complete loss of enzyme activity. dCMP can protect the enzyme against NEM inactivation, but the dihydrofolate analogues methotrexate and aminopterin alone do not afford similar protection. Compared to dCMP alone, dCMP plus either methotrexate or aminopterin greatly enhances protection against NEM inactivation. DTNB inactivation is reversed by dithiothreitol. For both reagents, inactivation kinetics obey second-order kinetics. NEM inactivation is pH dependent with a pKa for a required thiol group of 9.15 +/- 0.11. Complete enzyme inactivation by both reagents involves the modification of one thiol group per mole of dimeric enzyme. There are two thiol groups in the totally denatured enzyme modified by either NEM or DTNB. Kinetic analysis of NEM inactivation cannot distinguish between these two groups; however, with DTNB kinetic analysis of 2-nitro-5-thiobenzoate release shows that enzyme inactivation is due to the modification of one fast-reacting thiol followed by the modification of a second group that reacts about 5-6-fold more slowly. In the presence of methotrexate, the stoichiometry of dCMP binding to the dimeric enzyme is 1:1 and depends upon a reduced thiol group. It appears that the two equally sized subunits are arranged asymmetrically, resulting in one thiol-containing active site per mole of dimeric enzyme.     

Chemical modification of Corynebacterium sarcosine oxidase: role of sulfhydryl and histidyl groups

Sarcosine oxidase [sarcosine: oxygen oxidoreductase (demethylating) EC 1.5.3.1] from Corynebacterium contained 8 sulfhydryl groups per mol of enzyme as determined with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the presence of 0.2% SDS and by titration with p-chloromercuribenzoate (PMB). Among them, 2 groups were easily modified by iodoacetamide (IAA) and the modification resulted in complete loss of enzymatic activity. The inactivation by IAA followed first-order kinetics with respect to IAA concentration. The presence of acetate, a competitive inhibitor (I), protected the enzyme from inactivation by IAA. However, the protection was only approximately 50%. The enzyme was also inactivated by PMB, but in this case, there was practically no recovery of activity after treatment with thiol compounds. The enzyme was also rapidly inactivated by incubation with diethylpyrocarbonate (DEP). The absorbance change accompanying the inactivation showed that a single histidyl residue was modified by DEP, resulting in a complete loss of enzymatic activity. In the presence of acetate, the enzyme was completely protected from DEP-inactivation. Furthermore, DEP-inactivated enzyme recovered its enzymatic activity on treatment with hydroxylamine. These observations seem to imply that the modified histidine is essential for enzyme activity. In addition, modification by DEP changed the absorption spectrum in the visible region. This strongly suggests that the modified histidyl residue is present in the vicinity of the flavin moiety of the enzyme molecule.