Effects of GroESL coexpression on the folding of nicotinoprotein formaldehyde dismutase from Pseudomonas putida F61

The overexpression of fdm, which encodes the formaldehyde dismutase from Pseudomonas putida F61, resulted in the formation of inclusion bodies made up of aggregated enzyme, leaving little activity in the soluble fraction of the transformant cells. On the other hand, coexpression of groESL along with fdm facilitated in vivo solubilization of the enzyme protein in its active form. When coexpressed with groESL, formaldehyde dismutase purified from E. coli had the same crystalline form (i.e., a regular octahedron) as the native enzyme, and like the native enzyme, it bound 1 mol of NAD(H) and 2 mol of zinc in each subunit.     

Chemical modification of the lysine residues of bacterial formate dehydrogenase

Inactivation of formate dehydrogenase by formaldehyde, pyridoxal and pyridoxal phosphate was studied. The effects of concentrations of the modifying agents, substrates, products and inhibitors on the extent of the enzyme inactivation were examined. A complete formate dehydrogenase inactivation by pyridoxal, pyridoxal, phosphate and formaldehyde is achieved by the blocking of 2, 5 and 13 lysine residues per enzyme subunit, respectively. The coenzymes do not protect formate dehydrogenase against inactivation. In the case of modification by pyridoxal and pyridoxal phosphate a complete maintenance of the enzyme activity and specific protection of one lysine residue per enzyme subunit is observed during formation of a binary formate-enzyme complex, or a ternary enzyme--NAD--azide complex. One lysine residue is supposed to be located at the formate-binding site of the formate dehydrogenase active center.     

Formaldehyde dehydrogenase from Pseudomonas putida. Purification and some properties

Formaldehyde dehydrogenase was isolated and purified in an overall yield of 12% from cell-free extract of Pseudomonas putida C-83 by chromatographies on columns of DEAE-cellulose, DEAE-Sephadex A-50, and hydroxyapatite. The purified enzyme was homogeneous as judged by disc gel electrophoresis and was most active at pH 7.8 using formaldehyde as a substrate. The enzyme was also active toward acetaldehyde, propionaldehyde, glyoxal, and pyruvaldehyde, though the reaction rates were low. The enzyme was NAD+-linked but did not require the external addition of glutathione, in contrast with the usual formaldehyde dehydrogenase from liver mitochondria, baker's yeast, and some bacteria. The enzyme was markedly inhibited by Ni2+, Pd2+, Hg2+, p-chloromercuribenzoate, and phenylmethanesulfonyl fluoride. The molecular weight of the enzyme was estimated to be 150,000 by the gel filtration method, and analysis by SDS-polyacrylamide gel electrophoresis indicated that the enzyme was composed of two subunit monomers. Kinetic analysis gave Km values of 67 microM for formaldehyde and 56 microM for NAD+, and suggested that the reaction proceeds by a "Ping-pong" mechanism. The enzyme catalyzed the oxidation of formaldehyde accompanied by the stoichiometric reduction of NAD+, but no reverse reaction was observed.     

Nonpolar interactions in the modification of an essential sulfhydryl of sorbitol dehydrogenase by N-alkylmaleimides

A series of N-alkylmaleimides varying in chainlength from N-methyl- to N-octylmaleimide inclusive was shown to effectively inactivate sheep liver sorbitol dehydrogenase at pH 7.5 and 25 degrees C. The apparent second-order rate constants for inactivation increased with increasing chainlength of the N-alkylmaleimide used. Positive chainlength effects were also indicated by the Kd values for the N-ethyl and N-heptyl derivatives obtained from studies of the saturation kinetics observed for inactivation of the enzyme at high concentrations of these maleimides. The complete inactivation of sorbitol dehydrogenase was demonstrated to occur through the selective covalent modification of one cysteine residue per subunit of enzyme. The stoichiometry of enzyme inactivation was supported on the one hand by fluorescence titration with fluorescein mercuric acetate of the native and the inactivated enzyme, and, on the other hand, by the simultaneous inactivation of the enzyme with selective modification of one sulfhydryl per subunit by N-[p-(2-benzoxazolyl)phenyl]maleimide. Protection of the enzyme from N-alkylmaleimide inactivation was observed with the binding of NADH, whereas both NAD and sorbitol were ineffective as protecting ligands. Diazotized 3-aminopyridine adenine dinucleotide, in contrast to previous studies of this reagent with yeast alcohol dehydrogenase and rabbit muscle glycerophosphate dehydrogenase, did not function as a site-labeling reagent for sorbitol dehydrogenase.     

The role of arginine residues in the function of D-glyceraldehyde-3-phosphate dehydrogenase.

Inactivation of apo-glyceraldehyde-3-phosphate dehydrogenase from rat skeletal muscle in the presence of butanedione is the result of modification of one arginyl residue per subunit of the tetrameric enzyme molecule. The loss of activity follows pseudo-first-order kinetics. NAD+ increases the apparent first-order rate constant of inactivation. The effect of NAD+ on the enzyme inactivation is cooperative (Hill coefficient = 2.3--3.2). Glyceraldehyde 3-phosphate protected the holoenzyme against inactivation, decreasing the rate constant of the reaction. At saturating concentrations of substrate the protection was complete. The Hill plot demonstrates that the effect is cooperative. This suggests that subunit interactions in the tetrameric holoenzyme molecule may affect the reactivity of the essential arginyl residues. In contrast, glyceraldehyde 3-phosphate had no effect on the rate of inactivation of the apoenzyme in the presence of butanedione. 100 mM inorganic phosphate protected both the apoenzyme and holoenzyme against inactivation. The involvement of the microenvironment of the arginyl residues in the functionally important conformational changes of the enzyme is discussed.     

Purification and properties of tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli.

The aroH gene of Escherichia coli, which encodes the tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase isoenzyme of the common aromatic biosynthetic pathway, was cloned behind the tac promoter in expression plasmid pKK223-3. The enzyme was overexpressed, purified to homogeneity, and characterized. The native enzyme was found to be a dimeric metalloprotein containing 0.3 mol of iron per mol of subunit and variable amounts of zinc. The activity of the native enzyme was stimulated two- to threefold when assayed in the presence of Fe2+ ions. Pretreatment of the enzyme with Fe2+ also resulted in activation, accompanied by an equivalent increase in iron content. Treatment of the enzyme with chelating agents led to inactivation, which was fully reversed by the presence of Fe2+ in the assay mixture. The native enzyme exhibited a unique absorption profile, having a shoulder of absorbance on the aromatic band with a maximum around 350 nm and a broad, weak band with a maximum around 500 nm. Treatment of the enzyme with Fe2+ enhanced the absorbance at 350 nm and eliminated the band at 500 nm. Treatment with reducing agents caused the disappearance of both bands and destabilized the enzyme. Feedback regulation of the activity of the enzyme was specific for tryptophan, with maximum inhibition at about 70%.     

Derivatives of the purple phosphatase from red kidney bean: Replacement of zinc with other divalent metal ions

Apoenzyme, containing ⩽0.1 zinc atoms and ⩽0.2 Fe atoms per subunit and with ⩽3% of the phosphatase activity, has been prepared from native red kidney bean purple phosphatase. Treatment of this apoenzyme with Fe³⁺ or Zn²⁺ separately gave very little recovery of activity, whereas treatment with both Fe³⁺ and Zn²⁺ resulted in complete restoration of activity, indicating that both metal ions are essential. ZnFe enzyme with close to one iron and one zinc atom per subunit has been reconstituted by this procedure. Essentially full reactivation was also achieved by addition of Fe³⁺ together with Fe²⁺ or Co²⁺ to the apoenzyme; Fe³⁺ and Cd²⁺ gave 27% restoration of activity, whereas Fe³⁺ with Mn²⁺, Cu²⁺, Ni²⁺ or Hg²⁺ gave little or no increase in activity. Kinetic parameters for the hydrolysis of p-nitrophenyl phosphate and ATP by the FeFe derivative are reported.     

pH-dependent changes of intrinsic fluorescence of chemically modified liver alcohol dehydrogenases.

Horse liver alcohol dehydrogenase specifically carboxymethylated on cysteine-46 (a ligand to the zinc in the active site) or acetimidylated on 25 of the 30 lysine residues per subunit (including residue 228) was studied. The tryptophan fluorescence of these enzymes decreased by 35% as pH was increased, with an apparent pKa of 9.8 +/- 0.2, identical with that of native enzyme. Native enzyme in the presence of 30mM-imidazole, which displaces a water molecule ligated to the zinc, also had a pKa of 9.8. The ionoizable group is thus neither the water molecule nor one of the modified groups. Binding of NAD+ shifted the pKa for the fluorescence transition to 7.6 with native enzyme and to 9.0 with acetimidylated enzyme, but did not shift the pKa of carboxymethylated enzyme. Binding of NAD+ and trifluoroethanol, an unreactive alcohol, gave maximal fluorescence quenching at pH7 with all three enzymes. The acetimidylated enzyme--NAD+--trifluoroethanol complex had an apparent pKa of 5.0, but the pK of the native enzyme complex was experimentally inaccessible. The results are interpreted in terms of coupled equilibria between two different conformational states. On binding of NAD+, the modified enzymes apparently change conformation less readily than does native enzyme, but binding of alcohol can drive the change to completion.     

NAD-linked, GSH- and factor-independent aldehyde dehydrogenase of the methylotrophic bacterium, Hyphomicrobium X

Cell-free extracts of Hyphomicrobium X showed NAD-dependent aldehyde dehydrogenase activity, provided that NAD addition preceded that of aldehyde. Activity was lost rather rapidly, especially during purification attempts, but this could be partially masked by including a time-dependent restoration step with thiol compounds in the protocol. The nature of the assay buffer appeared to be critical and stimulation occurred on incorporation of K+ ions in the mixture. An even higher specific activity could be achieved by 1,4-dithiothreitol (DTT) treatment of the preparation, followed by removal of DTT, and assaying in the absence of thiol compounds under anaerobic conditions. Exposure of such a preparation to O2 led to a significant decrease in activity within a couple of hours. Immediate inactivation occurred on addition of H2O2, but this could be prevented completely by prior addition of NAD. Since GSH does not participate in the reaction and no stimulating factor was detected, the role of thiol compounds is most probably confined to restoration or prevention of damage to an O2-sensitive, necessary thiol group. Since the same features were found for cell-free extract as for the partially purified enzyme, only one enzyme type seems to be present. Although the enzyme is a general aldehyde dehydrogenase, the kinetic parameters and the specific activity of the cell-free extract for formaldehyde indicate that it may play a role in formaldehyde dissimilation by Hyphomicrobium X. The NAD-linked, GSH- and factor-independent aldehyde dehydrogenase described here appears to be different in several respects from the formaldehyde dehydrogenase of Pseudomonas putida (EC 1.2.1.46) (despite showing similar behavior toward coenzymes and factors) but resembles the aldehyde dehydrogenase from baker's yeast (EC 1.2.1.5).     

Modification of pig M4 lactate dehydrogenase by pyridoxal 5''-phosphate. Demonstration of an essential lysine residue.

1. Pig M4 lactate dehydrogenase treated in the dark with pyridoxal 5'-phosphate at pH8.5 and 25 degrees C loses activity gradually. The maximum inactivation was 66%, and this did not increase with concentrations of pyridoxal 5'-phosphate above 1 mM. 2. Inactivation may be reversed by dialysis or made permanent by reducing the enzyme with NaBH4. 3. Spectral evidence indicates modification of lysine residues, and 6-N-pyridoxyl-lysine is present in the hydrolsate of inactivated, reduced enzyme. 4. A second cycle of treatment with pyridoxal 5'-phosphate and NaBH4 further decreases activity. After three cycles only 9% of the original activity remains. 5. Apparent Km values for lactate and NAD+ are unaltered in the partially inactivated enzyme. 6. These results suggest that the covalently modified enzyme is inactive; failure to achieve complete inactivation in a single treatment is due to the reversibility of Schiff-base formation and to the consequent presence of active non-covalently bonded enzyme-modifier complex in the equilibrium mixture. 7. Although several lysine residues per subunit are modified, only one appears to be essential for activity: pyruvate and NAD+ together (both 5mM) completely protect against inactivation, and there is a one-to-one relationship between enzyme protection and decreased lysine modification. 8. NAD+ or NADH alone gives only partial protection. Substrates give virtually none. 9. Pig H4 lactate dehydrogenase is also inactivated by pyridoxal 5'-phosphate. 10. The possible role of the essential lysine residue is discussed.     

Inactivation of Escherichia coli L-threonine dehydrogenase by 2,3-butanedione. Evidence for a catalytically essential arginine residue

Incubation of homogeneous preparations of L-threonine dehydrogenase from Escherichia coli with 2,3-butanedione, 2,3-pentanedione, phenylglyoxal, or 1,2-cyclohexanedione causes a time- and concentration-dependent loss of enzymatic activity; plots of log percent activity remaining versus time are linear to greater than 90% inactivation, indicative of pseudo-first order inactivation kinetics. The reaction order with respect to the concentration of modifying reagent is approximately 1.0 in each case suggesting that the loss of catalytic activity is due to one molecule of modifier reacting with each active unit of enzyme. Controls establish that this inactivation is not due to modifier-induced dissociation or photoinduced nonspecific alteration of the dehydrogenase. Essentially the same Km but decreased Vmax values are obtained when partially inactivated enzyme is compared with native. NADH (25 mM) and NAD+ (70 mM) give full protection against inactivation whereas much higher concentrations (i.e. 150 mM) of L-threonine or L-threonine amide provide a maximum of 80-85% protection. Amino acid analyses coupled with quantitative sulfhydryl group determinations show that enzyme inactivated 95% by 2,3-butanedione loses 7.5 arginine residues (out of 16 total)/enzyme subunit with no significant change in other amino acid residues. In contrast, only 2.4 arginine residues/subunit are modified in the presence of 80 mM NAD+. Analysis of the course of modification and inactivation by the statistical method of Tsou (Tsou, C.-L. (1962) Sci. Sin. 11, 1535-1558) demonstrates that inactivation of threonine dehydrogenase correlates with the loss of 1 "essential" arginine residue/subunit which quite likely is located in the NAD+/NADH binding site.     

The sulfhydryl content of L-threonine dehydrogenase from Escherichia coli K-12: relation to catalytic activity and Mn2+ activation

When oxidized to cysteic acid by performic acid or converted to carboxymethylcysteine by alkylation of the reduced enzyme with iodoacetate, a total of six half-cystine residues/subunit are found in L-threonine dehydrogenase (L-threonine: NAD+ oxidoreductase, EC 1.1.1.103; L-threonine + NAD(+)----2-amino-3-oxobutyrate + NADH) from Escherichia coli K-12. Of this total, two exist in disulfide linkage, whereas four are titratable under denaturing conditions by dithiodipyridine, 5,5'-dithiobis(2-nitrobenzoic acid), or p-mercuribenzoate. The kinetics of enzyme inactivation and of modification by the latter two reagents indicate that threonine dehydrogenase has no free thiols that selectively react with bulky compounds. While incubation of the enzyme with a large excess of iodoacetamide causes less than 10% loss of activity, the native dehydrogenase is uniquely reactive with and completely inactivated by iodoacetate. The rate of carboxymethylation by iodoacetate of one -SH group/subunit is identical with the rate of inactivation and the carboxymethylated enzyme is no longer able to bind Mn2+. NADH (0.5 mM) provides 40% protection against this inactivation; 60 to 70% protection is seen in the presence of saturating levels of NADH plus L-threonine. Such results coupled with an analysis of the kinetics of inactivation caused by iodoacetate are interpreted as indicating the inhibitor first forms a reversible complex with a positively charged moiety in or near the microenvironment of a reactive -SH group in the enzyme before irreversible alkylation occurs. Specific alkylation of one -SH group/enzyme subunit apparently causes protein conformational changes that entail a loss of catalytic activity and the ability to bind Mn2+.     

Horse liver alcohol dehydrogenase. A study of the essential lysine residue.

1. The inactivation of horse liver alcohol dehydrogenase by pyridoxal 5'-phosphate in phosphate buffer, pH8, at 10 degrees C was investigated. Activity declines to a minimum value determined by the pyridoxal 5'-phosphate concentration. The maximum inactivation in a single treatment is 75%. This limit appears to be set by the ratio of the first-order rate constants for interconversion of inactive covalently modified enzyme and a readily dissociable non-covalent enzyme-modifier complex. 2. Reactivation was virtually complete on 150-fold dilution: first-order analysis yielded an estimate of the rate constant (0.164min-1), which was then used in the kinetic analysis of the forward inactivation reaction. This provided estimates for the rate constant for conversion of non-covalent complex into inactive enzyme (0.465 min-1) and the dissociation constant of the non-covalent complex (2.8 mM). From the two first-order constants, the minimum attainable activity in a single cycle of treatment may be calculated as 24.5%, very close to the observed value. 3. Successive cycles of modification followed by reduction with NaBH4 each decreased activity by the same fraction, so that three cycles with 3.6 mM-pyridoxal 5'-phosphate decreased specific activity to about 1% of the original value. The absorption spectrum of the enzyme thus treated indicated incorporation of 2-3 mol of pyridoxal 5'-phosphate per mol of subunit, covalently bonded to lysine residues. 4. NAD+ and NADH protected the enzyme completely against inactivation by pyridoxal 5'-phosphate, but ethanol and acetaldehyde were without effect. 5. Pyridoxal 5'-phosphate used as an inhibitor in steady-state experiments, rather than as an inactivator, was non-competitive with respect to both NADH and acetaldehyde. 6. The partially modified enzyme (74% inactive) showed unaltered apparent Km values for NAD+ and ethanol, indicating that modified enzyme is completely inactive, and that the residual activity is due to enzyme that has not been covalently modified. 7. Activation by methylation with formaldehyde was confirmed, but this treatment does not prevent subsequent inactivation with pyridoxal 5'-phosphate. Presumably different lysine residues are involved. 8. It is likely that the essential lysine residue modified by pyridoxal 5'-phosphate is involved either in binding the coenzymes or in the catalytic step. 9. Less detailed studies of yeast alcohol dehydrogenase suggest that this enzyme also possesses an essential lysine residue.     

Characterization of S1 nuclease. Involvement of carboxylate groups in metal binding.

Modification of the carboxylate groups of purified S1 nuclease resulted in a loss of its single-stranded DNAase, RNAase and phosphomonoesterase activities. The inactivation was due to the removal of zinc atoms from the enzyme and this in turn was dependent on the degree of modification. While the removal of one zinc atom resulted in the partial inactivation of the enzyme, removal of the remaining zinc atoms resulted in the complete inactivation of the enzyme. Similar results were obtained when the purified enzyme was incubated with various concentrations of the metal chelator, EDTA. The EDTA-(1 mM)-treated enzyme, depleted of one zinc atom, showing 40-45% residual activity, when incubated with 1 mM Zn2+ or 1 mM Co2+, regained a significant amount of its initial activity towards all the substrates. However, Woodward's-Reagent-K-modified enzyme depleted of one zinc atom and having the same level of activity (40-45%) could not regain its activity, indicating that the carboxylate groups are involved in the metal binding. Data obtained with carboxylate-group modification, EDTA-treatment, reconstitution with metal ions, zinc estimation and CD analysis of the enzyme suggests that, out of three zinc atoms present in S1 nuclease, zinc I is easily replaceable and is probably involved in the catalytic activity while zinc II and zinc III are involved in maintaining the enzyme structure.     

Effect of Carbohydrates and Starvation on Nitrogen Sparing Action of Methionine and Threonine in Rats

Formaldehyde dehydrogenase from Pseudomonas putida C-83 was found to contain 7 halfcystine residues per subunit monomer, as checked by the method of performic acid oxidation. Approximately 7 sulfhydryl groups per subunit monomer were titrated with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) after denaturation with 8 m urea. In the native enzyme, modification of three sulfhydryl groups per subunit with p-chloromercuribenzoate (PCMB) led to the complete loss of enzyme actiyities for both formaldehyde and n-butanol. Hydrogen-peroxide competitively inhibited the enzyme activity for formaldehyde, while it was only slightly inhibitory to the activity for n-butanol. Both formaldehyde and hydrogen-peroxide protected one sulfhydryl group per subunit monomer from modification with PCMB. Moreover, hydrogen-peroxide was hardly reactive to the enzyme which was preincubated with formaldehyde.

From these observations, we conclude that one of three PCMB-reactive sulfhydryl groups is essential for the binding of formaldehyde, and hydrogen-peroxide modifies this sulfhydryl group.     

Presence and quantity of dehydroalanine in histidine ammonia-lyase from Pseudomonas putida

Dehydroalanine is present in the histidine ammonia-lyase (histidase) from Pseudomonas putida ATCC 12633 as shown by reaction of purified enzyme with K14CN or NaB3H4 and subsequent identification of [14C]aspartate or [3H]alanine, respectively, following acid hydrolysis of the labeled protein. When labeling with cyanide was conducted under denaturing conditions, 4 mol of [14C]cyanide was incorporated per mol of enzyme (Mr 220 000), equivalent to one dehydroalanine residue being modified per subunit in this protein composed of four essentially identical subunits. In native enzyme, inactivation of catalytic activity by cyanide was complete when 1 mol of [14C]cyanide had reacted per mol of histidase, suggesting that modification of any one of the four dehydroalanine residues in the tetrameric enzyme was sufficient to prevent catalysis at all sites. Loss of activity on treatment with cyanide could be blocked by the addition of the competitive inhibitor cysteine or substrate if Mn2+ was also present. Cross-linking of native enzyme with dimethyl suberimidate produced no species larger than tetramer, thereby eliminating the possibility that an aggregation phenomenon might explain why only one-fourth of the dehydroalanyl residues was modified by cyanide during inactivation. A labeled tryptic peptide was isolated from enzyme inactivated with [14C]cyanide. Its composition was different from that of a tryptic peptide previously isolated from other histidases and shown to contain a highly reactive and catalytically important cysteine residue. Such a finding indicates the dehydroalanine group is distinct from the active site cysteine. Treatment of crude extracts with [14C]cyanide and purification of the inactive enzyme yielded labeled protein that release [14C]aspartate on acid hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immobilized flounder muscle glyceraldehyde 3-phosphate dehydrogenase

Summary Partially purified flounder muscle (Pseudopleuronectus americanus) glyceraldehyde 3-phosphate dehydrogenase was immobilized on cyanogen bromide-activated Sepharose. The catalytic properties of the immobilized preparation were studied to determine if immobilization alters the kinetic properties of the native holoenzyme. The results indicate that the pH activity profile of immobilized glyceraldehyde 3-phosphate dehydrogenase did not differ from that of the native enzyme. The Michaelis constants (Km) for NAD and glyceraldehyde 3-phosphate were somewhat altered. The enzyme stability toward various inactivation treatments in the presence and absence of NAD was characterized and compared to that of he native enzyme. When either form of the enzyme was incubated with urea at concentrations greater than 2m, inactivation occurred very rapidly. Incubation in 0.1% trypsin for 60 minutes decreased the activity of immobilized glyceraldehyde 3-phosphate dehydrogenase by 45% and of the native soluble enzyme by 70%. The immobilized enzyme also exhibited considerably more stability than the native soluble enzyme when exposed to a temperature of 50° or to 20 mm ATP. In all cases NAD either greatly reduced the rate of inactivation or completely protected the enzyme from inactivation.     

Brain succinic semialdehyde dehydrogenase. Reactions of sulfhydryl residues connected with catalytic activity.

Incubation of an NAD+-dependent succinic semialdehyde dehydrogenase from bovine brain with 4-dimethylaminoazobenzene-4-iodoacetamide (DABIA) resulted in a time-dependent loss of enzymatic activity. This inactivation followed pseudo first-order kinetics with a second-order rate constant of 168 m(-1).min(-1). The spectrum of DABIA-labeled enzyme showed a characteristic peak of the DABIA alkylated sulfhydryl group chromophore at 436 nm, which was absent from the spectrum of the native enzyme. A linear relationship was observed between DABIA binding and the loss of enzyme activity, which extrapolates to a stoichiometry of 8.0 mol DABIA derivatives per mol enzyme tetramer. This inactivation was prevented by preincubating the enzyme with substrate, succinic semialdehyde, but not by preincubating with coenzyme NAD+. After tryptic digestion of the enzyme modified with DABIA, two peptides absorbing at 436 nm were isolated by reverse-phase HPLC. The amino acid sequences of the DABIA-labeled peptides were VCSNQFLVQR and EVGEAICTDPLVSK, respectively. These sites are identical to the putative active site sequences of other brain succinic semialdehyde dehydrogenases. These results suggest that the catalytic function of succinic semialdehyde dehydrogenase is inhibited by the specific binding of DABIA to a cysteine residue at or near its active site.     

Biochemical properties of rat liver mitochondrial aldehyde dehydrogenase with respect to oxidation of formaldehyde.

The oxidation of formaldehyde by rat liver mitochondria in the presence of 50 mM phosphate was enhanced 2-fold by exogenous NAD+. Absolute requirement of NAD+ for formaldehyde oxidation was demonstrated by depleting the mitochondria of their NAD+ content (4.6 nmol/mg of protein), followed by reincorporation of the NAD+ into the depleted mitochondria. Aldehyde (formaldehyde) dehydrogenase activity was completely abolished in the depleted mitochondria, but the enzyme activity was restored to control levels following reincorporation of the pyridine nucleotide. Phosphate stimulation of formaldehyde oxidation could not be explained fully by the phosphate-induced swelling which enhances membrane permeability to NAD+, since stimulation of the enzyme activity by increased phosphate concentrations was still observed in the absence of exogenous NAD+. The Km for formaldehyde oxidation by the mitochondria was found to be 0.38 nM, a value similar to that obtained with varying concentrations of NAD+; both Vmax values were very similar, giving a value of 70 to 80 nmol/min/mg of protein. The pH optimum for the mitochondrial enzyme was 8.0. Inhibition of the enzyme activity by anaerobiosis was apparently due to the inability of the respiratory chain to oxidize the generated NADH. The inhibition of mitochondrial formaldehyde oxidation by succinate was found to be due to a lowering of the NAD+ level in the mitochondria. Succinate also inhibited acetaldehyde oxidation by the mitochondria. Malonate, a competitive inhibitor of succinic dehydrogenase, blocked the inhibitory effect of succinate. The respiratory chain inhibitors, rotenone, and antimycin A plus succinate, strongly inhibited formaldehyde oxidation by apparently the same mechanism, although the crude enzyme preparation (freed from the membrane) was slightly sensitive to rotenone. The mitochondria were subfractionated, and 85% of the enzyme activity was found in the inner membrane fraction (mitoplast). Furthermore, separation into inner membrane and matrix components indicated a distribution of aldehyde dehydrogenase activity similar to malic dehydrogenase.     

Formate dehydrogenase. Subunit and mechanism of inhibition by cyanide and azide.

Formate dehydrogenase (EC 1.2.1.2) prepared from peas (Pisum sativum) was a two-subunit enzyme. The enzyme accelerated the formation of an NAD+-cyanide compound having an adsorption band at 330 nm. The enzyme was able to bind one NAD+ molecule per each subunit but only 1 mole of NAD+-cyanide compound was formed per two subunits. The complex of NAD+, cyanide, and the enzyme was very stable and had no catalytic activity. Azide inhibited the formate dehydrogenase reaction in two different ways. By incubation of the enzyme with azide in the presence of NAD+, half of its catalytic activity was lost. The remaining activity was also inhibited by azide but this inhibition was removed competively by formate. Contrary to the case of cyanide the inhibition by azide could be removed by dialysis and no spectral species due to the addition compound of NAD+ and azide could be observed. The data from double recipricol plots of the initial velocity and the formate concentration led to a conclusion that formate dehydrogenase has two sites with about equal catalytic activity. The Km for formate was different for the two catalytic sites (1.67 and 6.25 mM) but the difference was not noticeable in the case of the Km for NAD+.