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This article presents information on forthcoming papers to be published in coming issues of the periodical "European Journal of Biochemistry." Some of the papers are: "Localization of Histone HS in the Subunit Organization of Chromatin Using ImmunoeIectron Microscopy"; "Presence of an Amino Acid Oxidase in Photosystem II of Anacystis nidutans"; "Aldoximes: Active-Site Probes of Alcohol Dehydrogenases"; and "Quinone Reductases of Higher Plants."

Amino acid analyses of L-lactate dehydrogenase from baker's show that the minimum molecular weight (53 000 daltons) of the protein is much lower than found in the literature (80 000). This result, combined with those reported in the following papers, leads to a revision of the dimeric model generally accepted for cytochrome b2 [ABSTRACT FROM AUTHOR]

The preceding paper in this journal has reported that pyruvate could be substituted for 2-oxoglutarate as a substrate of saccharopine dehydrogenase [ε-N-(L-glutaryl-2)-L-lysine:NAD oxidoreductase (L-lysine-forming)] in the direction of reductive condensation. In the present communication, the kinetic mechanism of saccharopine dehydrogenase reaction with NADH, L-lysine and pyruvate as reactants is reported. The results of initial velocity study, inhibition studies with lysine analogs and a reaction product, NAD+M, are consistent with an ordered mechanism with the coenzyme binding first and pyruvate last. The reaction mechanism is at variance with that of the normal reaction in which 2-oxoglutarate is the substrate, in that the order of addition of the amino and oxo acid substrates is reversed. This fact suggests that there exists a small degree of randomness in the binding of amino and oxo acid substrates. From a product inhibition study, NAD+M was shown to be the last reactant released. Saccharopine [ε-N-(L-glutaryl-2)-L-lysine] was found to act as a potent dead-end inhibitor of the condensation reactions (of lysine and 2-oxoglutarate, and of lysine and pyruvate) by forming an abortive E · NADH · saccharopine complex. [ABSTRACT FROM AUTHOR]

1. The amino acid sequence of D-glyceraldehyde-3-phosphate dehydrogenase from the extreme thermophile Thermus aquaticus has been elucidated. 2. The polypeptide contains 332 amino acids and its sequence is 70% identical with that of the enzyme from the moderate thermophile Bacillus stearothermophilus. 3. In contrast to less thermostable forms of the enzymes from B. stearothermophilus, pig, lobster and yeast, the T. aquaticus enzyme has only one cysteine residue, namely cysteine-149 which is required for catalysis. [ABSTRACT FROM AUTHOR]

Amino acid analyses of L-lactate dehydrogenase from baker's show that the minimum molecular weight (53 000 daltons) of the protein is much lower than found in the literature (80 000). This result, combined with those reported in the following papers, leads to a revision of the dimeric model generally accepted for cytochrome b2 [ABSTRACT FROM AUTHOR]

A putative phosphoglycerate dehydrogenase (PGDH), which catalyzes the oxidation of d-phosphoglycerate to 3-phosphohydroxypyruvate in the so-called phosphorylated serine metabolic pathway, from the enteric protozoan parasite Entamoeba histolytica was characterized. The E. histolytica PGDH gene ( EhPGDH) encodes a protein of 299 amino acids with a calculated molecular mass of 33.5 kDa and an isoelectric point of 8.11. EhPGDH showed high homology to PGDH from bacteroides and another enteric protozoan ciliate, Entodinium caudatum. EhPGDH lacks both the carboxyl-terminal serine binding domain and the 13–14 amino acid regions containing the conserved Trp139 (of Escherichia coli PGDH) in the nucleotide binding domain shown to be crucial for tetramerization, which are present in other organisms including higher eukaryotes. EhPGDH catalyzed reduction of phosphohydroxypyruvate to phosphoglycerate utilizing NADH and, less efficiently, NADPH; EhPGDH did not utilize 2-oxoglutarate. Kinetic parameters of EhPGDH were similar to those of mammalian PGDH, for example the preference of NADH cofactor, substrate specificities and salt-reversible substrate inhibition. In contrast to PGDH from bacteria, plants and mammals, the EhPGDH protein is present as a homodimer as demonstrated by gel filtration chromatography. The E. histolytica lysate contained PGDH activity of 26 nmol NADH utilized per min per mg of lysate protein in the reverse direction, which consisted 0.2–0.4% of a total soluble protein. Altogether, this parasite represents a unique unicellular protist that possesses both phosphorylated and nonphosphorylated serine metabolic pathways, reinforcing the biological importance of serine metabolism in this organism. Amino acid sequence comparison and phylogenetic analysis of various PGDH sequences showed that E. histolytica forms a highly supported monophyletic group with another enteric protozoa, cilliate E. caudatum, and bacteroides. [ABSTRACT FROM AUTHOR]

Five residues involved in catalysis and coenzyme binding have been identified in D-2-hydroxy-4-methylvalerate dehydrogenase from Lactobacillus delbrueckii subsp. bulgaricus by using biochemical and genetical methods. Enzyme inactivation with diethylpyrocarbonate indicated that a single histidine residue was involved in catalysis. Since H296 is the only conserved histidine in the whole family of NAD-dependent D-2-hydroxyacid dehydrogenases, we constructed the H296Q and H296S mutants and showed that their catalytic efficiencies were reduced 105-fold compared with the wild-type enzyme. This low residual activity was shown to be insensitive to diethylpyrocarbonate. Taken together these data demonstrate that H296 is responsible for proton exchange in the redox reaction. Two acidic residues (D259 and E264) were candidates for maintaining H296 in the protonated state and their roles were examined by mutagenesis. The D259N and E264Q mutant enzymes both showed similar and large reductions in their kcat Km ratios (200–800-fold, depending on pH), indicating that either D259 or E264 (or both) could partner H296. The conserved R235 residue was a candidate for binding the α-carboxyl group of the substrate and it was changed to lysine. The R235K mutant showed a 104-fold reduced kcat Km due to both an increased Km and a reduced kcat for 2-oxo-4-methylvalerate. Thus R235 plays a role in binding the substrate carboxylate similar to R171 in the L-lactate dehydrogenases. Finally, we constructed the H205Q mutant to test the role of this partially conserved histidine residue (in 10/13 enzymes of the family). This mutant enzyme displayed a 7.7-fold increased Kcat and a doubled catalytic efficiency at pH 5, was as sensitive to diethylpyrocarbonate as the wild-type but showed a sevenfold increased Km for NADH and a 100-fold increase in Kd for NADH together with 10–30-fold lower substrate inhibition. The transient kinetic behaviour of the H205Q mutant is as predicted from our previous study on the enzymatic mechanism of D-2-hydroxy-4-methylvalerate dehydrogenase which showed that coenzyme binding is highly pH dependent and indicated that release of the oxidised coenzyme is a significant component of the rate-limiting processes in catalysis at pH 6.5. [ABSTRACT FROM AUTHOR]

Three amino acid residues in the active site of lipoamide dehydrogenase from Azotobacter vinelandii were replaced with other residues. His450, the active-site base, was replaced with Scr, Tyr or Phe. Pro451, from X-ray analysis found to be in cis conformation positioning the backbone carbonyl of His450 close to N3 of the flavin, was changed to Ala. Glu455, from X-ray analysis expected to be involved in modulating the pKa of the base (His450), was replaced with Asp and Gln. The general conclusion is that mutation of the His-Glu diad impairs intramolecular electron transfer between the disulfide/dithiol and the FADH¯/FAD. The wild-type enzyme functions according to a ping-pong mechanism in the physiological reaction in which the formation o1' NADH is rate-limiting. Above pH 8.0 the enzyme is strongly inhibited by the product NADH. The pH dependence of the steady-state kinetics using the NAD+ analog 3-acetylpyridine adenine dinucleotide (AcPyAde+) reveals a pKa of 8.1 in the pKm AcPyAde+ plot indicating that this pKa is related to the deprotonation of His450 [Benen, J., Berkel van, W., Zak, Z., Visser. T., Veeger, C, & Kok de, A. (1991) Eur. J. Biochem. 202, 863–872] and to the inhibition by NADH. The mutations considerably affect turnover, Enzymes with the mutations Pro451→Ala, His450→Phe and His450→Tyr appear to be almost inactive in both directions. Enzyme His450→Ser is minimally active, V at the pH optimum being 0.5% of wild-type activity in the physiological reaction. Rapid reaction kinetics show that lot the His450-mutated enzymes the reductive half reaction using reduced 6,8-thioctic acid amide [Lip(SH)2] is rate-limiting and extremely slow when compared to the wild-type enzyme. For enzyme Pro451→Ala it is concluded that the loss of activity is due to over-reduction by Lip(SH)2 and NADH. The Glu4SS-mutated enzymes arc catalytically competent but show strong inhibition by the product NADH (enzyme Glu455→Asp more than Glu455→Gln). The inhibition can largely be: overcome by using AcPyAde+ instead of NAD+ in the physiological reaction. The rapid reaction kinetics obtained for enzymes Glu455→Asp and Glu455→Gln deviate from the wild-type enzyme. It is concluded that this difference is due to cooperativity between the active sites in this dimeric enzyme. Rapid reaction kinetics of enzymes His450→Ser and Glu455→Gln show the existence of two intermediates at the two-electron reduced level: a species with the NAD+ bound, the flavin reduced and the disulfide intact (oxidized) and a species with NAD+ bound, the disulfide reduced and the flavin oxidized. No spectral evidence is obtained for the participation of a proposed flavin C4a adduct intermediate [Thorpe, C. & Williams, C, H. Jr (1981) Biochemistry 20, 1507–1513] in the reaction mechanism of enzymes His450→Ser and Glu455→Gln. [ABSTRACT FROM AUTHOR]

Three amino acid residues in the active site of lipoamide dehydrogenase from Azotobacter vinelandii were replaced by other residues. His450, the active-site base, was changed into Ser, Tyr and Phe. Pro451, in cis conformation, was changed into Ala. Glu455 was replaced with Asp and Gin. Absorption, fluorescence and CD spectroscopy of the mutated enzymes in their oxidized state (Eox) showed only minor changes with respect to the wild-type enzyme, whereas considerable changes were observed in the spectra of the two-electron-reduced (EH2) species of the enzymes upon reduction by the substrate dihydrolipoamide. Differences in extent of reduction of the flavin by NADH indicate that the redox potential of the flavin is altered by the mutations. Enzyme Pro455→Ala showed the greatest deviation from wild type. The enzyme is very easily over-reduced to the four-electron reduced state (EH4) by dihydrolipoamide. This is probably due to a change in the backbone conformation caused by the cis-trans conversion. From studies on the pH dependence of the thiolate charge-transfer absorption and the relative fluorescence of EH2 of the enzymes, it is concluded that mutation of His450 results in a relatively simple and easily interpreted distribution of electronic species at the EH2 level. For all three His450-mutated enzymes an apparent pKa1 near 5.5 is calculated that is assigned to the interchange thiol. A second apparent pKa2 is calculated of 6.9, 7.5 and 7.1 for the His450→Phe, -Ser and -Tyr enzymes, respectively, and signifies the deprotonation of the tautomeric equilibrium between the interchange and charge-transfer thiols. The difference in apparent pKa2 values between the His450-mutated enzymes is explained by changes in micropolarity. At the EH2 level the wild-type enzyme consists of multiple electronic forms as reported for the Escherichia coli enzyme [Wilkinson, K. D. and Williams C. H. Jr (1979) J. Biol. Chem. 254, 852–862]. Based on the results obtained with the His450-mutated enzymes, it is concluded that the lowest pKa is associated with the interchange thiol. A model for the equilibrium species of the wild-type enzyme at the EH2 level is presented which takes three pKa values into account. The results of the pH dependence of the electronic species at the EH2 level of Glu455-mutated enzymes essentially follow the model proposed for the wild-type enzyme. However mutation of Glu455 shifts the tautomeric equilibrium of EH2 in favor of the charge-transfer species. This effect is explained by impaired ion-pair formation between the protonated His and the interchange thiolate and a lowering of the pKa of His450. [ABSTRACT FROM AUTHOR]

The tetrameric enzyme D-glyceraldehyde-3-phosphate dehydrogenase from the moderate thermophile Bacillus stearothermophilus is more stable to thermal denaturation than its counterpart from lobster muscle [Harris et al. (1980) Eur. J. Biochem. 108, 535-547]. Extra buried ionic bonds between subunits of the thermophilic enzyme make an important contribution to thermal stabilisation. Further stabilisation of the tetrameric enzyme is derived from additional hydrophobic interactions between the S-loops at the core of the tetramer. In the enzyme from the extreme thermophile Thermus aquaticus, which is even more thermostable, intersubunit ion pairs must also play a role but changes in interactions at the surface appear to be equally important. Thus additional hydrophobic interactions at the edge of subunit interfaces would prevent access of water to the interior of the molecule. Furthermore, the arrangement of charged residues on the surface of the T. aquaticus enzyme would allow maximal surface ion pair formation. The presence of surface ion pairs in other proteins correlates well with thermal stability [Perutz, M. F. and Raidt, H. (1975) Nature (Lond.) 255, 256-258] and would provide a general stabilising influence on the subunit in this case. [ABSTRACT FROM AUTHOR]

Cross-linking of the unimer of glutamate dehydrogenase from beef liver (consisting of six polypeptide chains each having a molecular weight of 56 000) with dimethyladipimidate and subsequent analysis by sodium dodecylsulfate electrophoresis shows predominantly the trimeric species (molecular weight 168 000). Treatment with dimethylimidates of other chain length yields significantly, less trimeric species indicating that the amino groups being cross-linked are within a distance of about 0.85 nm. Comparison of the molar amount of incorporated [14C]dimethyladipimidate with the number of modified amino groups (determined with trinitrobenzenesulfonic acid) shows that although 8-9 of the 34 amino groups have reacted, only 2-3 of them are involved in cross-links. Reaction with dimethylimidates inactivates the enzyme. The loss of the activity is partly concomitant to cross-linking to the trimeric species and not simply due to the modification of essential lysine residues. This is supported by the fact that, although more lysine residues react with monofunctional methylimidates, the loss of activity is reduced. Purified chymotryptic and tryptic peptides of the radioactive-labeled trimeric species were subjected to sequence analysis. Six peptides containing 75% of the total label were identified: one involves the amino-terminal residue alanine-1 and the others involve lysine-105, lysine-154, lysine-269, lysine-358 and lysine-399. Quantitative analysis of the specific radioactivity of each peptide mol lysine leads to the conclusion that only lysine-105, lysine-154, lysine-269 and lysine-358 participate in cross-links, lysine-269 and lysine-358, respectively, being at isologous and lysine-105 cross-linked with lysine-154 at heterologous contact domains of the enzyme. A model for the planar arrangement of the trimeric species in the quaternary structure of glutamate dehydrogenase is discussed. It includes both isologous and heterologous contact areas between the polypeptide chains. [ABSTRACT FROM AUTHOR]

1. D-Gtyceraldehyde-3-phosphate dehydrogenase fi'om an extreme thermophile, T. thermophilus strain HB8, was purified and crystallized. 2. Tile enzyme was found to possess remarkable heat stability, being slowly inactivated at 90°C. 3. Basic kinetic constants and pH profile are reported. The enzyme was activated 25-fold by 90 mM NH4Cl, and also by ethanol up to 5-fold at 30°C. 4. The enzyme was found to be far more resistant to urea or sodium dodecylsulfate than the rabbit enzyme. 5. The enzyme was shown to be a tetramer of molecular weight 130000–135000. Amino acid composition analysis revealed no unusual features. Circular dichroic spectra suggested that the contents of the ordered structure of the thermophile enzyme are similar to those of the rabbit enzyme. 6. The other catalytic properties of the thermophile enzyme are discussed in comparison with those of the enzymes from other sources. [ABSTRACT FROM AUTHOR]

We have studied the denaturation difference spectra of glyceraldehyde-3-phosphate dehydrogenase and lactic dehydrogenase in the 270–300mμ and 220–250mμ wavelength regions. When hydrogen ion concentration is varied in the range from pH 7 to pH 2, the absorption changes at 230–232 mμ are very similar to those at 291–293mμ with both dehydrogenases. The pH dependence of the optical rotatory parameters bo and [m′]232 follows a very similar pattern. The addition of sodium dodecyl sulfate induces hardly any change in the optical rotatory properties of the two dehydrogenases, while the difference spectra both in the near and in the far ultraviolet region closely resemble the corresponding denaturation difference spectra. The solvent perturbation spectra of aromatic chromophores due to 20% ethylene glycol exhibit a maximum in the far ultraviolet region at about 227mμ. At this wavelength, the extent of perturbation of tryptophan is 6.3 times greater than that of tyrosine. Calculations based on the far ultraviolet perturbation of aromatic chromophores suggest that the far ultraviolet denaturation difference spectra of the two dehydrogenases may be attributed to changes in the environment of aromatic chromophores, particularly of tryptophan. [ABSTRACT FROM AUTHOR]