Carboxylic Acid Substrates Research Articles

Substrate specificity of acetyl coenzyme A synthetase.

Acetyl coenzyme A synthetase (EC 6.2.1.1) has been examined for its ability to accept various carboxylic acids as substrates in place of acetic acid. The activity of the enzyme with these substrates was monitored using a coupled enzyme assay and high pressure liquid chromatography (HPLC) analysis. Short chain carboxylic acids were found to be active including: propionic, acrylic, fluoroacetic, methacrylic, 3-chloropropionic, 3-bromopropionic, and propiolic. The kinetic parameters, Km and % Vmax of the carboxylic acid substrates, are reported and show that these acids are poorer substrates than acetic acid. Several of the acyl CoAs were synthesized on a preparative scale using enzyme catalysis, purified using preparative HPLC, and characterized using proton NMR spectroscopy. In the course of the NMR identification, a complete and fully resolved spectral assignment for all the protons of coenzyme A was made and is reported. The acyl-CoA analogs should be useful as substrate analogs and as potential affinity labels for enzymes that bind acetyl-CoA.

Inactivation of D-(-)-beta-hydroxybutyrate dehydrogenase by modifiers of carboxyl and histidyl groups.

Data presented in this paper suggest that D-(-)-beta-hydroxybutyrate dehydrogenase (BDH) purified from bovine heart mitochondria contains an essential carboxyl group and an essential histidyl residue at or near the active site. Lactate and malate dehydrogenases, which catalyze reactions analogous to that catalyzed by BDH, also contain an aspartyl and a histidyl residue at the active site [Birktoft, J.J., & Banaszak, L.J. (1983) J. Biol. Chem. 258, 472-482]. In addition, all three enzymes contain an essential arginyl residue, apparently concerned with electrostatic interaction with their respective carboxylic acid substrates, and promote ternary adduct formation involving the enzyme, NAD, and sulfite.

Reaction of pyridoxal 5′-phosphate with Escherichia coli CoA transferase: Evidence for an essential lysine residue

The acetyl-CoA:acetoacetate CoA-transferase of Escherichia coli was reversibly inactivated by pyridoxal 5′-phosphate. The residual activity of the enzyme was dependent on the concentration of the modifying reagent to a concentration of 5 m m. The maximum level of inactivation was 89%. Kinetic and equilibrium analyses of inactivation were consistent with a two-step process (Chen and Engel, 1975, Biochem. J. 149, 619) in which the extent of inactivation was limited by the ratio of first-order rate constants for the reversible formation of an inactive Schiff base of pyridoxal 5′-phosphate and the enzyme from a noncovalent, dissociable complex of the enzyme and modifier. The calculated minimum residual activity was in close agreement with the experimentally determined value. The conclusion that the loss of catalytic activity resulted from modification of a lysine residue at the active site was based on the following data, (a) After incubation with 5 m m pyridoxal 5′-phosphate, 3.95 mol of the reagent was incorporated per mole of free enzyme with 89% loss of activity, while 2.75 mol of pyridoxal 5′-phosphate was incorporated into the enzyme-CoA intermediate with a loss of 10% of catalytic activity; the intermediate was formed in the presence of acetoacetyl-CoA; (b) acid hydrolysis of the modified, reduced enzyme-CoA intermediate yielded a single fluorescent compound that was identified as N 6-pyridoxyllysine by chromatography in two solvent systems; (c) the enzyme was also protected from inactivation by saturating concentrations of free CoA and ADP but not by adenosine. The results suggested that a lysine residue is involved in the electrostatic binding of the pyrophosphate group of CoA. Carboxylic acid substrate did not protect the enzyme from inactivation.

Mechanism of pigeon liver malic enzyme: kinetics, specificity, and half-site stoichiometry of the alkylation of a cysteinyl residue by the substrate-inhibitor bromopyruvate.

Malic enzyme from pigeon liver is alkylated by the substrate analogue bromopyruvate, resulting in the concomitant loss of its oxidative decarboxylase and oxalacetate decarboxylase activities, but not its ability to reduce alpha-keto acids. The inactivation of oxidative decarboxylase activity follows saturation kinetics, indicating the formation of an enzyme-bromopyruvate complex (K congruent to 8 mM) prior to alkylation. The inactivation is inhibited by metal ions and pyridine nucleotide cofactors. Protection of malic enzyme by the substrates L-malate and pyruvate and the inhibitors tartronate and oxalate requires the presence of the above cofactors, which tighten the binding of these carboxylic acids in accord with the ordered kinetic scheme (Hsu, R. Y., Lardy, H. A., and Cleland, W. W. (1967), J. Biol. Chem. 242, 5315-5322). Bromopyruvate is reduced to L-bromolactate by malic enzyme and is an effective inhibitor of L-malate and pyruvate in the overall reaction. The apparent kinetic constants (90 muM-0.8 mM) are one to two orders of magnitude lower than the half-saturation constant (K) of inactivation, indicating a similar tightening of bromopyruvate binding in the E-NADP+ (NADPH)-Mn2+ (Mg2+)-BP complexes. During alkylation, bromopyruvate interacts initially at the carboxylic acid substrate pocket of the active site, as indicated by the protective effect of substrates and the ability of this compound to form kinetically viable complexes with malic enzyme, particularly as a competitive inhibitor of pyruvate carboxylation with a Ki (90 muM) in the same order as its apparent Michaelis constant of 98 muM. Subsequent alkylation of a cysteinyl residue blocks the C-C bond cleavage step. The incorporation of radioactivity from [14C]bromopyruvate gives a half-site stoichiometry of two carboxyketomethyl residues per tetramer, indicating strong negative cooperativity between the four subunits of equal size, or alternatively the presence of structurally dissimilar active sites.

Mechanism of malic enzyme from pigeon liver. Magnetic resonance and kinetic studies of the role of Mn2+.

As determined by EPR, malic enzyme from pigeon liver binds Mn2+ with a half-site stoichiometry of two tight binding sites (KD=6 to 10 mum) per enzyme tetramer and at two to four weak binding sites (KD=0.43 to 1.34 mM). The activation of malic enzyme by Mn2+ at high levels of L-malate shows biphasic kinetics yielding two activator constants for Mn2+. The dissociation constants of Mn2+ for both classes of sites are of the same order as the kinetically determined activator constants of Mn2+, indicating active site binding at both classes of binding sites. The binding of Mn2+ to the tight sites enhances the paramagnetic effect of Mn2+ on 1/T1 of water protons by a factor (epsilon) of 17, while binding at the weak sites yields a smaller epsilon of 11. The coenzymes TPN and TPNH have no effects on epsilon, while the carboxylic acid substrates L-malate and pyruvate and the inhibitors D-malate and oxalate significantly decrease epsilon. TPNH causes a 38-fold tightening of binding of the substrate L-malate to the enzyme-Mn2+ complex, consistent with the previously described highly ordered kinetic scheme, but only a 2-fold tightening of binding of the competitive inhibitor D-malate. The dissociation constant of L-malate from the quaternary E-Mn2+-TPNH-L-malate complex (32 muM) agrees with the Km of L-malate (25 muM), indicating active site binding. The dissociation constants of pyruvate from the ternary E-Mn2+-pyruvate complex (12 mM) and from the quaternary E-Mn2+-TPN-pyruvate complex (20 mM) are similar to the Km of pyruvate (5 mM), also indicating active site binding and a less highly ordered kinetic scheme for the reactions of pyruvate than for those of L-malate. Analysis of the frequency dependence of 1/T1 of water protons indicates that two fast exchanging water ligands remain coordinated to Mn2+ in the binary E-Mn2+ complex. The binding of the substrates L-malate and pyruvate and of the transition state analog oxalate to the E-Mn2+ complex decrease the number of fast exchanging water ligands on Mn2+ by approximately 1, but the binding of D-malate has no significant effect on this parameter, indicating the occlusion or replacement of a water ligand of the enzyme-bound Mn2+ by a properly oriented substituent on C-2 of the substrate. Occlusion rather than replacement of a water ligand by pyruvate is established by studies of 1/T1 of 13COO- and 13CO-enriched pyruvate which indicate second sphere Mn2+ to pyruvate distances of 4.6 A (COO-) and 4.8 A (CO) in the ternary enzyme-Mn2+-pyruvate complex. Formation of the quaternary complex with TPN increases these distances by 0.8 A, indicating the participation of a second sphere enzyme-Mn2+-(H2O)-pyruvate complex in catalysis. Thus, malic enzyme, like five other enzymes which utilize metals to polarize carbonyl groups, forms a second sphere complex with its substrate.

Purification and properties of Escherichia coli coenzyme A-transferase

The acetyl-CoA:acetoacetate-CoA-transferase has been purified 36-fold to homogeneity from an acetoacetate degradation operon ( ato) constitutive mutant of Escherichia coli. The enzyme has the following physical properties: Stokes radius, 40.5 Å; diffusion coefficient ( D 20,w), 5.32 × 10 −7 cm s −1; sedimentation coefficient ( s 20,w), 5.38S; molecular weight, 97,000 and a frictional ratio ( f f 0 ) of 1.35. The enzyme is composed of two α subunits ( M r = 26,000) and two β subunits ( M r = 23,000). E. coli CoA-transferase contains six cysteine residues per mole of enzyme and no disulfide bonds. The native transferase reacts with 4 mol of p-chloromercuribenzoate per 97,000 g of enzyme. Two cysteine residues react rapidly with p-chloromercuribenzoate resulting in an 85% inactivation of enzyme activity. The reactivity of these two residues is enhanced at least fivefold in the presence of acetyl-CoA. Acetoacetate has no effect on the rate of reaction of p-chloromercuribenzoate with the enzyme. E. coli CoA-transferase is partially inactivated by acyl-CoA substrates in the absence of carboxylic acid substrates, presumably as the result of a metal-catalyzed acylation of the ϵ-amino group of a lysine residue near the active site. The enzyme utilizes a variety of short chain acyl-CoA and carboxylic acid substrates but exhibits maximal activity with normal and 3-keto substrates.

Mechanism of Pigeon Liver Malic Enzyme. Modification of Sulfhydryl Groups by 5,5′-Dithiobis(2-nitrobenzoic Acid) and N-Ethylmaleimide

Abstract Malic enzyme of pigeon liver (l-malate:NADP+ oxidoreductase (decarboxylating) EC 1.1.1.40) contains a total of approximately 10 —SH groups per subunit, reactive with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Two or more exposed groups (Class I) are highly reactive with DTNB; alteration of these groups does not impair the oxidative decarboxylase activity. This activity, however, is lost upon reaction of a less accessible —SH group (Class II), the alteration of which also results in concomitant increase in the ability of this enzyme to reduce pyruvate in the presence of NADPH. The remaining —SH groups (Class III) in the undenatured enzyme are largely unreactive with this reagent. The alkylating reagent N-ethylmaleimide (NEM) induced similar changes in the over-all and reductase activities of this enzyme. These effects by NEM are also attributed to the alteration of Class II —SH group, since they are prevented by reversible attachment of a thionitrobenzoate substituent (from DTNB) to this —SH group prior to reaction with NEM. Modification of Class II —SH group by either reagent is promoted by carboxylic acid substrates, and partially prevented by Mn2+. This group appears to be located at or near the active center of this enzyme, but is not directly involved in the binding of reactants nor in the catalytic process. An —SCN derivative was prepared by reacting the DTNB-modified enzyme with KCN. This derivative was found to be completely active in oxidative decarboxylation. Apparently, the geometry of the active center is such that this activity is impaired by the attachment of a relatively bulky or charged substituent to the Class II —SH group. Furthermore, our experimental data implicate that modification results in the blockage of the reversible C—C bond cleavage step, thereby accounting for the loss of oxidative decarboxylase and decarboxylase activities.