Effects of changing glutamate 487 to lysine in rat and human liver mitochondrial aldehyde dehydrogenase. A model to study human (Oriental type) class 2 aldehyde dehydrogenase

Many Oriental people possess a liver mitochondrial aldehyde dehydrogenase where glutamate at position 487 has been replaced by a lysine, and they have very low levels of mitochondrial aldehyde dehydrogenase activity. To investigate the cause of the lack of activity of this aldehyde dehydrogenase, we mutated residue 487 of rat and human liver mitochondrial aldehyde dehydrogenase to a lysine and expressed the mutant and native enzyme forms in Escherichia coli. Both rat and human recombinant aldehyde dehydrogenases showed the same molecular and kinetic properties as the enzyme isolated from liver mitochondria. The E487K mutants were found to be active but possessed altered kinetic properties when compared to the glutamate enzyme. The Km for NAD+ at pH 7.4 increased more than 150-fold, whereas kcat decreased 2-10-fold with respect to the recombinant native enzymes. Detailed steady-state kinetic analysis showed that the binding of NAD+ to the mutant enzyme was impaired, and it could be calculated that this resulted in a decreased nucleophilicity of the active site cysteine residue. The rate-limiting step for the rat E487K mutant was also different from that of the recombinant rat liver aldehyde dehydrogenase in that no pre-steady-state burst of NADH formation was found with the mutant enzyme. Both the rat native enzyme and the E487K mutant oxidized chloroacetaldehyde twice as fast as acetaldehyde, indicating that the rate-limiting step was not hydride transfer or coenzyme dissociation but depended upon nucleophilic attack. Each enzyme form showed a 2-fold activation upon the addition of Mg2+ ions. Substituting a glutamine for the glutamate did not grossly affect the properties of the enzyme. Glutamate 487 may interact directly with the positive nicotinamide ring of NAD+ for the Ki of NADH was the same in the lysine enzyme as it was in the glutamate form. Because of the altered NAD+ binding properties and kcat of the E487K variant, it is assumed that people possessing this form will not have a functional mitochondrial aldehyde dehydrogenase.     

Horse liver alcohol dehydrogenase-catalyzed oxidation of aldehydes: dismutation precedes net production of reduced nicotinamide adenine dinucleotide

The oxidation of aldehydes by horse liver alcohol dehydrogenase (HL-ADH) is more complex than previously recognized. At low enzyme concentrations and/or high aldehyde concentrations, a pronounced lag in the assay progress curve is observed when the reaction is monitored for NADH production at 340 nm. When the progress of the reaction is followed by 1H NMR spectroscopy, rapid dismutation of the aldehyde substrate into the corresponding acid and alcohol is observed during the lag phase. Steady-state production of NADH commences only after aldehyde concentrations drop below 5% of their initial value; thereafter, NADH production occurs with continuous adjustment of the equilibrium between aldehyde, alcohol, NADH, and NAD+. The steady-state NADH production exhibits normal Michaelis-Menten kinetics and is in accord with earlier studies using much higher enzyme concentrations where no lag phase was reported. These results establish that the ability of HL-ADH to oxidize aldehydes is much greater than previously thought. The relationship between aldehyde dismutase and aldehyde dehydrogenase activities of HL-ADH is discussed.     

Identification of the subunit and important target peptides of pig heart NAD-dependent isocitrate dehydrogenase modified by the affinity label adenosine 5'-O-[S-(4-bromo-2, 3-dioxobutyl)thiophosphate]

Pig heart NAD-dependent isocitrate dehydrogenase is inactivated by adenosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)thiophosphate] (AMPS-BDB) with incorporation of 1.78 mol of reagent/mol of average subunit. Complete protection against the inactivation is provided by 20 mM isocitrate + 1 mM Mn2+, and the incorporation is decreased to about 1.3 mol of reagent/mol of average subunit. The addition of NAD, NADH, or Mn2+ alone has little effect on the functional changes produced by AMPS-BDB, while ADP gives only partial protection against the inactivation. The ability of ADP to decrease the Km for isocitrate is not affected by the AMPS-BDB modification of the enzyme. These results indicate that the isocitrate substrate site is the target of AMPS-BDB. The enzyme has three types of subunits with a tetramer having the composition alpha2 beta gamma. Here, [2-3H]AMPS-BDB-modified subunits are separated by HPLC on a C4 reverse-phase column, after the treatment of the modified enzyme with 4 M urea. The predominant radioactivity is distributed in alpha and gamma subunits. However, evidence based on recombination of subunits from modified and unmodified enzymes indicates that only labeling of the alpha subunit is responsible for inactivation by AMPS-BDB. Subsequently, the separated modified subunits were chemically cleaved by CNBr and then purified by HPLC using a C18 column. The labeled peptides were further digested by pepsin, purified by HPLC, and sequenced. These results indicate that R88 and R98 from the alpha subunit are the major targets of AMPS-BDB which cause inactivation and that these are at or near the isocitrate site of the enzyme.     

Adoption of beta structure by the inactivating "ball" peptide of the Shaker B potassium channel

The conformation of the inactivating peptide of the Shaker B K+ channel (ShB peptide) and that of a noninactivating mutant (ShBL7E peptide) have been studied. Under all experimental conditions explored, the mutant peptide remains in a predominantly nonordered conformation. On the contrary, the inactivating ShB peptide has a great tendency to adopt a highly stable beta structure, particularly when challenged "in vitro" by anionic phospholipid vesicles. Because the putative peptide binding elements at the inner mouth of the channel comprise a ring of anionic residues and a hydrophobic pocket, we hypothesize that the conformational restrictions imposed on the ShB peptide by its interaction with the anionic lipid vesicles could partly imitate those imposed by the above ion channel elements. Thus, we propose that adoption of beta structure by the inactivating peptide may also occur during channel inactivation. Moreover, the difficulties encountered by the noninactivating ShBL7E peptide mutant to adopt beta structure and the observation that trypsin hydrolysis of the ShB peptide prevent both structure formation and channel inactivation lend further support to the hypothesis that adoption of beta structure by the inactivating peptide in a hydrophobic environment is important in determining channel blockade.     

Histidine decarboxylase of Lactobacillus 30a: inactivation and active-site labeling by L-histidine methyl ester

Histidine decarboxylase from Lactobacillus 30a is rapidly and irreversibly inactivated upon incubation with L-histidine methyl ester. The rate of inactivation is first-order with respect to remaining active enzyme and exhibits saturation kinetics with a kinact of 1.2 mM and an apparent first-order rate constant of 0.346 min-1 at pH 4.8 and 25 degrees C. On complete inactivation, 3 mol of [14C]histidine (from L-[14C]histidine methyl ester) and 2 mol of 14C (from L-histidine [14C]methyl ester) are bound in nondialyzable form per mol (190 000 g) of protein inactivated with a corresponding loss of three of the five DTNB-titratable--SH groups that are essential for activity of the native enzyme. Imidazole propionate, a competitive inhibitor of the enzyme, protects against inactivation, loss of --SH groups, and incorporation of radioactivity from both the histidine and the methyl ester moieties of the labeled inhibitor, and kinetic evidence indicates that imidazole propionate and histidine methyl ester compete for binding at the active site of histidine decarboxylase in a mutually exclusive manner. Treatment of the labeled protein with either alkali or hydroxylamine results in the quantitative release of radioactivity. These data suggest that inactivation of histidine decarboxylase by L-histidine methyl ester results from two different modes of interaction between the inhibitor and the active site of histidine decarboxylase; the major interaction involves an essential -SH group.     

Identification of the benzomorphan opiate binding site on the catalytic subunit of acetylcholinesterase

The interaction of a benzomorphan opiate with the active site of the catalytic subunit of acetylcholinesterase was studied using photoaffinity labeling. UV irradiation of (-)-N-[3H]allylnormetazocine bound to Torpedo acetylcholinesterase resulted in covalent incorporation of 60-70% of the bound ligand. The labeled catalytic subunit was subjected to chemical cleavage with cyanogen bromide and proteolytic degradation with trypsin, chymotrypsin, and staphylococcal V8 protease. The resulting peptide fragments were purified by high performance liquid chromatography and sequenced in the gas phase. The label was not stable under the conditions of the sequencing, but a peptide fragment consisting of Gln74 to Glu82 was reproducibly labeled. These amino acids are located at the rim of a gorge leading to the active site of the enzyme. Molecular modeling studies then demonstrated that these residues can be placed within van der Waals contact of the (-)-N-[3H]allylnormetazocine molecule while it is bound to the active site of the enzyme.     

Involvement of conserved glycine residues, 229 and 234, of Vibrio harveyi aldehyde dehydrogenase in activity and nucleotide binding

The involvement of two conserved glycine residues (Gly229 and Gly234) in activity and nucleotide binding in Vibrio harveyi aldehyde dehydrogenase (ALDH) have been investigated. Each of the glycine residues has been mutated to alanine and the mutant ALDHs have been expressed in Escherichia coli and specifically labelled with [35S]methionine. The G229A mutant was inactive with either NADP+ or NAD+ as coenzyme and did not bind to 2',5'-ADP Sepharose, indicating a complete loss of nucleotide affinity. In contrast, the G234A mutant showed a high affinity for 2',5'-ADP Sepharose. Purified G234A mutant showed similar kinetic properties to the native enzyme including a pre-steady-state burst of NADPH; however, the Michaelis constants for NAD+ and NADP+ were increased by 3- to 9-fold, showing that the mutation had an effect on saturation of the enzyme with NAD(P)+. These data are consistent with the structure for the nucleotide binding domain of Vh.ALDH being similar to that of class 3 or class 2 mammalian ALDHs which differ from the classical nucleotide binding domain found in most dehydrogenases.     

Modulation of Na+ channel inactivation by the beta 1 subunit: a deletion analysis

Na+ currents recorded from Xenopus oocytes expressing the Na+ channel alpha subunit alone inactivate with two exponential components. The slow component predominates in monomeric channels, while co-expression with the beta 1 subunit favors the fast component. Macropatch recordings show that the relative rates of these components are much greater than previously estimated from two-electrode measurements (approximately 30-fold vs approximately 5-fold). A re-assessment of steady-state inactivation, h infinity (V), shows that there is no depolarized shift of the slow component, provided a sufficiently long prepulse duration and repetition interval are used to achieve steady-state entry and recovery from inactivation, respectively. Deletion mutagenesis of the beta 1 subunit was used to define which regions of the subunit are required to modulate inactivation kinetics. The carboxy tail, comprising the entire predicted intracellular domain, can be deleted without a loss of activity; whereas small deletions in the extracellular amino domain or the signal peptide totally disrupt function.     

Effects of NAD or NADP on the stability of liver and pectoral muscle enzymes in 3-acetylpyridine treated quail by heat and trypsin

(1) The effects of long term treatment with 3-acetylpyridine on the stability of enzymes towards heat and trypsin treatment were studied. (2) In the liver NAD or NADP provided a similar degree of protection against heat inactivation at 55 degrees C for 6-phosphogluconate dehydrogenase (24%), glyceraldehyde-3-phosphate dehydrogenase (24%) and malic enzyme (20%), low level of protection of lactate dehydrogenase (13%) but didn't affect acetylcholinesterase at all. In the muscle, however, there was substantial protection against heat inactivation by coenzyme of glyceraldehyde-3-phosphate dehydrogenase (52%), an intermediate level of protection of lactate dehydrogenase (25%), low level of protection of 6-phosphogluconate dehydrogenase (17%) and malic enzyme (17%) and almost no protection of acetylcholinesterase. (3) In the susceptibility towards trypsin a low but similar degree of protection for dehydrogenases by coenzymes was observed in the liver whereas in the muscle there was substantial protection against trypsin inactivation by NAD of glyceraldehyde-3-phosphate dehydrogenase, an intermediate level of protection of 6-phosphogluconate dehydrogenase and malic enzyme and very little protection of lactate dehydrogenase but no protection of acetylcholinesterase. Among enzymes tested, glyceraldehyde-3-phosphate dehydrogenase showed the greatest protection against heat and trypsin inactivation by NAD. (4) The results suggest that the effect of 3-acetylpyridine treatment on the stability of muscle glyceraldehyde-3-phosphate dehydrogenase appears to be quite specific and selective.     

Identification of adenine binding domain peptides of the NADP+ active site within porcine heart NADP(+)-dependent isocitrate dehydrogenase

Photoaffinity labeling with [2'-32P]2N3NADP+ and [32P]2N3NAD+ was used to identify two overlapping tryptic and chymotryptic generated peptides within the adenine binding domain of NADP(+)-dependent isocitrate dehydrogenase (IDH). Photolysis was required for insertion of radiolabel, and prior photolysis of photoprobes before addition of IDH prevented insertion. Photoincorportion of 2N3NAD+ inhibited the enzymatic activity of IDH. Photolabeling of IDH with both [32P]2N3NAD+ and [2'-32P]2N3-NADP+ showed saturation effects with apparent Kds of 20 and 14 microM (+/-12%), respectively. The efficiency of photoincorporation at saturation of binding sites was determined to be about 50%. Also, photolabeling was observed with [32P]8N3ATP and [32P]2N3ATP but with saturation effects observed at lower affinity. With all radiolabeled probes reduction of photoinsertion was effected best by the addition of NADP+ followed by NAD+ and then ATP, indicating that photoinsertion with all the probes was within the NADP+ binding site. Isolation of [32P]2N3NAD+ and [2'-32P]2N3NADP+ photolabeled peptides by use of immobilized boronate and immobilized Al3+ chromatography, respectively, followed by HPLC purification resulted in the identification of overlapping peptides corresponding to Ile244-Arg249 and Leu121-Arg133 (tryptic fragments) and Lys243-His248 and Leu121-His135 (chymotryptic fragments). Trp125 and Trp245 were identified as the sites of photoinsertion based on these residues not being detectable on sequencing, the lack of chymotryptic cleavage at these residues, and the decreased rate of trypsin digestion at nearby Lys243 and Lys127. Sequence analysis of [32P]8N3ATP and [32P]2N3ATP photolabeled peptides gave essentially the same peptide regions being photolabeled but at much lower efficiency, indicating that the effects of ATP on IDH activity are dependent on competition for the same site.     

Identification of the nonsubstrate steroid binding site of rat liver glutathione S-transferase, isozyme 1-1, by the steroid affinity label, 3beta-(iodoacetoxy)dehydroisoandrosterone

3beta-(Iodoacetoxy)dehydroisoandrosterone (3beta-IDA), an analogue of the electrophilic substrate, Delta5-androstene-3,17-dione, as well as an analogue of several other steroid inhibitors of glutathione S-transferase, was tested as an affinity label of rat liver glutathione S-transferase, isozyme 1-1. A time-dependent loss of enzyme activity is observed upon incubation of 3beta-IDA with the enzyme. The rate of enzyme inactivation exhibits a nonlinear dependence on 3beta-IDA concentration, yielding an apparent Ki of 21 microM. Upon complete inactivation of the enzyme, a reagent incorporation of approximately 1 mol/mol of enzyme subunit or 2 mol/mol of enzyme dimer is observed. Protection against inactivation and incorporation is afforded by alkyl glutathione derivatives and nonsubstrate steroid ligands such as 17beta-estradiol-3,17-disulfate but, surprisingly, not by Delta5-androstene-3,17-dione or any other electrophilic substrate analogues tested. These results suggest that the site of reaction is within the nonsubstrate steroid binding site of the enzyme, which is distinguishable from the electrophilic substrate binding site, near the active site of the enzyme. Two cysteine residues, Cys17 and Cys111, are modified in nearly equal amounts, despite an average reagent incorporation of 1 mol/mol enzyme subunit. Isolation of enzyme subunits indicates the presence of unmodified, singly labeled, and doubly labeled subunits, consistent with mutually exclusive modification of cysteine residues across enzyme subunits; i.e., modification of Cys111 on subunit A prevents modification of Cys111 on subunit B and similarly for Cys17. Molecular modeling analysis suggests that Cys17 and Cys111 are located in the nonsubstrate steroid binding site, within the cleft between the subunits of the dimeric enzyme.     

A study of the alkaline mesentericopeptidase active site by means of peptide chloromethyl ketones

The kinetics of inactivation of alkaline mesentericopeptidase was studied using chloromethyl ketone derivatives of amino acids and peptides. It was shown that Tos-LysCH2Cl and Tos-PheCH2Cl did not influence the enzyme activity, while the inhibitory effect of Cbz-Ala-Gly-PheCH2Cl was 35 times that of Cbz-Ala-PheCH2Cl. The dependence of the pseudo-first order rate constant of the enzyme inactivation by Cbz-Ala-Gly-PheCH2Cl on pH and temperature indicated that a group with a pK of 6.59 and deltaHi of 7.7 kcal/mol was the site of the inhibitor's attack. Amino acid analysis of the modified totally inactive enzyme revealed a definite loss of histidine and after performic acid oxidation a recovery of 3-carboxymethyl histidine. The whole set of experimental data is convincing evidence on behalf of a selective alkylation of the N-3 of the active site histidine after treatment with Cbz-Ala-PheCH2Cl and Cbz-Ala-Gly-PheCH2Cl. Alkaline mesenteriocopeptidase possesses an extended active site and only a peptide chloromethyl ketone, covering a determined sequence of theenzyme molecule (S3, S2, S1, S'1, S'2, S'3 ...) is able to provide effective inhibition. The values of the inactivation constant (kinact) for Cbz-Ala-PheCH2Cl and Cbz-Ala-Gly-PheCH2Cl are close to the corresponding values reported for subtilisin Amylosacchariticus.     

Active site modifications in a double mutant of liver alcohol dehydrogenase: structural studies of two enzyme-ligand complexes

The oxidation of alcohol to aldehyde by horse liver alcohol dehydrogenase (LADH) requires the transfer of a hydride ion from the alcohol substrate to the cofactor nicotinamide adenine dinucleotide (NAD). A quantum mechanical tunneling contribution to this hydride transfer step has been demonstrated in a number of LADH mutants designed to enhance or diminish this effect [Bahnson, B. J., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 12797-12802]. The active site double mutant Phe93 --> Trp/Val203 --> Ala shows a 75-fold reduction in catalytic efficiency relative to that of the native enzyme, and reduced tunneling relative to that of either single mutant. We present here two crystal structures of the double mutant: a 2.0 A complex with NAD and the substrate analogue trifluoroethanol and a 2.6 A complex with the isosteric NAD analogue CPAD and ethanol. Changes at the active site observed in both complexes are consistent with reduced activity and tunneling. The NAD-trifluoroethanol complex crystallizes in the closed conformation characteristic of the active enzyme. However, the NAD nicotinamide ring rotates away from the substrate, toward the space vacated by replacement of Val203 with the smaller alanine. Replacement of Phe93 with the larger tryptophan also produces unfavorable steric contacts with the nicotinamide carboxamide group, potentially destabilizing hydrogen bonds required to maintain the closed conformation. These contacts are relieved in the second complex by rotation of the CPAD pyridine ring into an unusual syn orientation. The resulting loss of the carboxamide hydrogen bonds produces an open conformation characteristic of the apoenzyme.     

Wild isolates of murine cytomegalovirus induce myocarditis and antibodies that cross-react with virus and cardiac myosin

The pyruvate dehydrogenase complex (PDC) plays a key role in the anaerobic metabolism of the parasitic nematode Ascaris suum. Two isoforms of the alpha-subunit of pyruvate dehydrogenase (E1) have been identified: alpha I is most abundant in anaerobic adult muscle and alpha II in aerobic larvae. Both isoforms have been expressed as alpha 2 beta 2 tetramers with a muscle-specific beta-subunit, purified to apparent homogeneity, reconstituted with E1-deficient adult A. suum muscle PDC, and assayed for PDC and E1 kinase activity. Recombinant alpha II is a poor substrate for the adult E1 kinase, but its stoichiometry of phosphorylation/inactivation is similar to that reported for the human E1. Initially, inactivation parallels the incorporation of about 1 mol 32P/mol E1 and at maximal phosphorylation about 2.4 32P/mol E1 is incorporated. In contrast, recombinant alpha I (r alpha I) is phosphorylated rapidly, and substantially more phosphorylation accompanies inactivation. To examine this altered pattern of phosphorylation, the two phosphorylation sites in each E1 alpha subunit of the r alpha I (site 1 and site 2) were changed either individually or together from Ser to Ala by site-directed mutagenesis. Site 1 was phosphorylated more rapidly than site 2, but the phosphorylation of either site resulted in inactivation, and the phosphorylation of only a single E1 alpha subunit of the tetramer was necessary for inactivation. However, both E1 alpha subunits of the tetramer were phosphorylated, based on the incorporation of about 3.5 mol 32P/mol E1 at maximal phosphorylation and the altered mobility of most of the E1 alpha subunits during SDS-PAGE. These observations suggest that the regulation of both E1 isoforms is modified to maintain PDC activity during the transition to anaerobiosis.     

The location of the carboxy-terminal region of gamma chains in fibrinogen and fibrin D domains

Elongated fibrinogen molecules are comprised of two outer "D" domains, each connected through a "coiled-coil" region to the central "E" domain. Fibrin forms following thrombin cleavage in the E domain and then undergoes intermolecular end-to-middle D:E domain associations that result in double-stranded fibrils. Factor XIIIa mediates crosslinking of the C-terminal regions of gamma chains in each D domain (the gammaXL site) by incorporating intermolecular epsilon-(gamma-glutamyl)lysine bonds between amine donor gamma406 lysine of one gamma chain and a glutamine acceptor at gamma398 or gamma399 of another. Several lines of evidence show that crosslinked gamma chains extend "transversely" between the strands of each fibril, but other data suggest instead that crosslinked gamma chains can only traverse end-to-end-aligned D domains within each strand. To examine this issue and determine the location of the gammaXL site in fibrinogen and assembled fibrin fibrils, we incorporated an amine donor, thioacetyl cadaverine, into glutamine acceptor sites in fibrinogen in the presence of XIIIa, and then labeled the thiol with a relatively small (0.8 nm diameter) electron dense gold cluster compound, undecagold monoaminopropyl maleimide (Au11). Fibrinogen was examined by scanning transmission electron microscopy to locate Au11-cadaverine-labeled gamma398/399 D domain sites. Seventy-nine percent of D domain Au11 clusters were situated in middle to proximal positions relative to the end of the molecule, with the remaining Au11 clusters in a distal position. In fibrin fibrils, D domain Au11 clusters were located in middle to proximal positions. These findings show that most C-terminal gamma chains in fibrinogen or fibrin are oriented toward the central domain and indicate that gammaXL sites in fibrils are situated predominantly between strands, suitably aligned for transverse crosslinking.     

Mechanisms of inhibition of aldehyde dehydrogenase by nitroxyl, the active metabolite of the alcohol deterrent agent cyanamide

Nitroxyl, produced in the bioactivation of the alcohol deterrent agent cyanamide, is a potent inhibitor of aldehyde dehydrogenase (AIDH); however, the mechanism of inhibition of AlDH by nitroxyl has not been described previously. Nitroxyl is also generated from Angeli's salt (Na2N2O3) at physiological pH, and, indeed, Angeli's salt inhibited yeast AlDH in a time- and concentration-dependent manner, with IC50 values under anaerobic conditions with and without NAD+ of 1.3 and 1.8 microM, respectively. Benzaldehyde, a substrate for AlDH, competitively blocked the inhibition of this enzyme by nitroxyl in the presence of NAD+, but not in its absence, in accord with the ordered mechanism of this reaction. The sulfhydryl reagents dithiothreitol (5 mM) and reduced glutathione (10 mM) completely blocked the inhibition of AlDH by Angeli's salt. These thiols were also able to partially restore activity to the nitroxyl-inhibited enzyme, the extent of reactivation being dependent on the pH at which the inactivation occurred. This pH dependency indicates the formation of two inhibited forms of the enzyme, with an irreversible form predominant at pH 7.5 and below, and a reversible form predominant at pH 8.5 and above. The reversible form of the inhibited enzyme is postulated to be an intra-subunit disulfide, while the irreversible form is postulated to be a sulfinamide. Both forms of the inhibited enzyme are derived via a common N-hydroxysulfenamide intermediate produced by the addition of nitroxyl to active site cysteine thiol(s).     

NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase from Thermoproteus tenax. The first identified archaeal member of the aldehyde dehydrogenase superfamily is a glycolytic enzyme with unusual regulatory properties

The hyperthermophilic archaeum Thermoproteus tenax possesses two glyceraldehyde-3-phosphate dehydrogenases differing in cosubstrate specificity and phosphate dependence of the catalyzed reaction. NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase catalyzes the phosphate-independent irreversible oxidation of D-glyceraldehyde 3-phosphate to 3-phosphoglycerate. The coding gene was cloned, sequenced, and expressed in Escherichia coli. Sequence comparisons showed no similarity to phosphorylating glyceraldehyde-3-phosphate dehydrogenases but revealed a relationship to aldehyde dehydrogenases, with the highest similarity to the subgroup of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenases. The activity of the enzyme is affected by a series of metabolites. All effectors tested influence the affinity of the enzyme for its cosubstrate NAD+. Whereas NADP(H), NADH, and ATP reduce the affinity for the cosubstrate, AMP, ADP, glucose 1-phosphate, and fructose 6-phosphate increase the affinity for NAD+. Additionally, most of the effectors investigated induce cooperativity of NAD+ binding. The irreversible catabolic oxidation of glyceraldehyde 3-phosphate, the control of the enzyme by energy charge of the cell, and the regulation by intermediates of glycolysis and glucan degradation identify the NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase as an integral constituent of glycolysis in T. tenax. Its regulatory properties substitute for those lacking in the reversible nonregulated pyrophosphate-dependent phosphofructokinase in this variant of the Embden-Meyerhof-Parnas pathway.     

Characterization of the binding site of the tripeptide intermediate D-Phenylalanyl L-prolyl-L-valine in gramicidin S biosynthesis

The tripeptide intermediate D-Phe-Pro-Val in the biosynthesis of gramicidin S was labeled by incorporation of either L-[14C]phenylalanine or L-[14C]valine in an in vitro biosynthetic assay. The gramicidin S synthetase 2-tripeptide complex was first digested with CNBr and subsequently by Staphylococcus aureus V8 protease. The active site peptide carrying the radioactively labeled tripeptide was isolated in pure form by reversed phase high performance liquid chromatography technology and analyzed by liquid phase sequencing, mass spectrometry, and amino acid analysis. It was demonstrated that D-Phe-Pro-Val is attached to the 4'-phosphopantetheine cofactor at the thiolation center for valine of gramicidin S synthetase 2. In this way the attachment site of a peptide intermediate in nonribosomal peptide biosynthesis was identified for the first time. Our results are in full agreement with the multiple carrier model of nonribosomal peptide biosynthesis (Stein, T., Vater, J., Kruft, V., Otto, A., Wittmann-Liebold, B., Franke, P., Panico, M., McDowell, R., and Morris, H. R. (1996) J. Biol. Chem. 271, 15426-15435), which predicts that the growing peptide chain in the elongation process should always be bound to the thiotemplate site specific for its C-terminal amino acid component.     

Identification of an NAD+ binding site of brain glutamate dehydrogenase isoproteins by photoaffinity labeling

Photoaffinity labeling with [32P]nicotinamide 2-azidoadenosine dinucleotide (2N3NAD+) was used to identify the NAD+ binding site within two types of glutamate dehydrogenase isoproteins (GDH I and GDH II) isolated from bovine brain. In the absence of photolysis, 2N3NAD+ is a substrate for the GDH isoproteins. When the enzymes were covalently modified by photolysis in the presence of saturating amounts of photoprobe, about 50% inhibition of the GDH activities was observed. Photoinsertion of probe was increased by GTP or glutarate and decreased by NAD+ or ADP. With the combination of immobilized boronate affinity chromatography and reversed-phase HPLC, photolabel-containing peptides generated with trypsin were isolated. This identified a portion of the adenine ring binding domain of GDH isoproteins as the region containing the sequence, CIAVGXSDGSIWNPDGIDPK for both GDH isoproteins, corresponding to Cys270 through Lys289 of the amino acid sequence of well known bovine liver GDH. The X indicates a position for which no phenylthiohydantoin-derivative could be assigned. The missing residue, however, can be designated as a photolabeled glutamate since the sequences including the glutamate residue in question have a complete identity with those of the other GDH species known. Photolabeling of these peptides was prevented by the presence of NAD+ during photolysis. These results demonstrate selectivity of the photoprobe for the NAD+ binding site and suggest that the peptide identified using the photoprobe is located in the NAD+ binding domain of the brain GDH isoproteins. Both amino acid sequencing and compositional analysis identified Glu275 as the site of photoinsertion.