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P C Engel

Publications and source records attributed to P C Engel.

At least 73 records · Page 4Linked to original sources
PubMed1994

Identification of the reactive cysteine in clostridial glutamate dehydrogenase by site-directed mutagenesis and proof that this residue is not strictly essential.

Cys320 of clostridial glutamate dehydrogenase, a residue close to the coenzyme binding site, has been replaced by serine. The mutant enzyme was successfully overproduced and purified by using the normal protocol for the wild-type enzyme and also behaved indistinguishably from wild-type enzyme on native and SDS-PAGE. The specific activity was significantly enhanced in assays at both pH 7 (+90%) and pH 8 (+38%). Detailed initial-rate kinetics revealed that at pH 7 this increase was mainly attributable to a higher maximum rate, since the Km values for both substrates were marginally increased. In the mutant enzyme the inactivating reaction with DTNB that characterizes the wild-type enzyme is completely eliminated. This proves that inactivation of the wild-type enzyme is due to modification of Cys320, that nevertheless Cys320 is not strictly essential for catalytic activity and that the remaining cysteine residue at position 144 is inaccessible to DTNB. Provision of an engineered subunit with a correct native structure but with its DTNB titre decreased from 1 to 0 mol/mol now offers a valuable tool for counting subunits in hybrid oligomers.

Base Sequence↗
PubMed1994

Activity staining of halophilic enzymes: substitution of salt with a zwitterion in non-denaturing electrophoresis.

Several NAD(P)(+)-dependent dehydrogenases were partially purified from Halobacterium halobium. When salt (2M (NH4)2SO4) was replaced with glycine betaine (4M or 6M), the zwitterion stabilised activity less completely than the salt. Nevertheless most of the enzyme activity still remained after 90h, e.g. 70% for malate dehydrogenase. This level of stabilisation permitted non-denaturing gel electrophoresis in 4M betaine after dialysis to replace salt. Coomassie Blue staining showed good separation of the proteins, and activity staining, hitherto impossible for halophilic enzymes, readily identified the individual dehydrogenase bands. Transfer of activity-stained gels to Coomassie staining solution halted background formazan staining and showed up activity and other protein bands in contrasting colours.

Ammonium Sulfate↗
PubMed1993

Evolution of substrate diversity in the superfamily of amino acid dehydrogenases. Prospects for rational chiral synthesis.

We have analysed the sequence homology between glutamate, leucine and phenylalanine dehydrogenases in the light of the solution of the structure of the glutamate dehydrogenase from Clostridium symbiosum. This analysis indicates that the elements of secondary structure comprising the core of the two domains in glutamate dehydrogenase are conserved in the other two enzymes. There is a striking conservation of the residues responsible for the recognition of the nicotinamide ring of the nucleotide cofactor and the backbone of the amino acid substrates. Furthermore, residues involved in a major conformational rearrangement on amino acid binding are preserved, as are those implicated in the catalytic chemistry. In contrast, the pattern of insertions/deletions between these enzymes is consistent with possible differences in quaternary structure. Differential substrate specificity between these enzymes is achieved by critical substitutions at the base of the binding pocket, which accommodates the side-chain of the amino acid substrate. This provides insights into the mutations necessary to produce new catalysts for the chiral synthesis of novel amino acids.

Amino Acid Oxidoreductases↗
PubMed1993

Is pyridoxal 5'-phosphate an affinity label for phosphate-binding sites in proteins?: The case of bovine glutamate dehydrogenase.

The effects of pyridoxal 5'-phosphate (PalP) on ox liver glutamate dehydrogenase (94% inactivation by 1.8 mM reagent at pH 7 and 25 degrees C) have been compared with those of three analogues, 5'-deoxypyridoxal (96% inactivation), pyridoxal 5'-sulphate (97%) and pyridoxal 5-methylsulphonate (94%), in order to establish whether PalP acts as an affinity label for this enzyme. Like PalP and unlike pyridoxal, which is a much less potent inactivator, none of the analogues has a free 5'-OH group to cyclize with the aldehyde function. The result with 5'-deoxypyridoxal shows that a negative charge, such as that of the phosphate group, is not required for efficient inactivation. With all four reagents, addition of an excess of cysteine or lysine led to 90-100% re-activation over 3-20 h. Dialysis also caused reactivation to a similar extent. A combination of 2.15 mM NADH, 1 mM GTP and 10 mM 2-oxoglutarate gave complete protection against PalP, but only partial protection against the analogues. 5'-Deoxypyridoxal still caused 20-25% inactivation in the presence of the protection mixture. Absorbance measurements after reduction with NaBH4 show the characteristic features of a reduced Schiff's base and allowed estimation of the extent of reaction. With all the reagents the protection mixture decreased incorporation by about 1 mol/mol, but levels of incorporation without protection varied from about 2 mol/mol for PalP up to about 5 mol/mol for 5'-deoxypyridoxal. The labelling at additional sites may explain the residual inactivation in the presence of potent protecting agents.

Affinity Labels↗
PubMed1993

The mechanism of substrate and coenzyme binding to clostridial glutamate dehydrogenase during oxidative deamination.

The binding of NAD+ and L-Glutamate to glutamate dehydrogenase (GDH) from Clostridium symbiosum has been investigated by stopped-flow fluorescence spectroscopy. The formation of the binary complexes produces little change in the protein fluorescence but formation of the ternary complex results in quenching of its fluorescence with a maximum value of 40%. This finding, coupled with the finding that a step prior to hydride transfer but subsequent to ternary complex formation is rate limiting, has enabled us to monitor the kinetics of ternary complex formation in detail. The ternary complex can be formed via the GDH-NAD+ or the GDH-L-Glu binary complexes, but the route via the GDH-NAD+ binary complex is the preferred pathway. The equilibrium and rate constants for the formation of the two binary complexes and the ternary complex formed via the two possible pathways have been determined. These studies have revealed an interaction between the coenzyme-binding site and the substrate-binding site, which lead to a decrease in the binding constant for the second substrate binding to the enzyme. The free energy coupling between the binary and ternary complexes is about 2.4-2.8 kJ.mol-1. We propose that there is a further isomerisation of the ternary complex, which is rate limiting for the steady-state turnover of the enzyme. Formation of this complex is characterised by an increased negative interaction, with a free energy coupling between these complexes of 6.3-11.6 kJ.mol-1.

Binding Sites↗
PubMed1993

Inhibition of glutamate dehydrogenase by covalent coenzyme-substrate adducts: a re-examination.

Covalent adducts of NAD+ with pyruvate and 2-oxoglutarate have been reported to inhibit differentially the activities of bovine glutamate dehydrogenase (GDH) towards these two oxoacid substrates, implying separate active sites. Thorough reinvestigation fails to confirm this finding, with the pyruvate adduct uniformly the more potent inhibitor of both substrate activities under several assay conditions. This suggests that bovine GDH provides amino acid dehydrogenation sites of one structural type only. Clostridial GDH, with a strong preference for oxoglutarate over pyruvate as substrate, is also more strongly inhibited by the pyruvate adduct in the oxoglutarate assay. These findings challenge the generality of the view that carbonyl substrates used in forming such adducts confer specificity for the corresponding substrate binding pocket in enzyme active sites.

Animals↗
PubMed1993

Intrinsic crotonase activity in a bacterial butyryl-CoA dehydrogenase.

Butyryl-CoA, crotonyl-CoA or 3-hydroxybutyryl-CoA all ultimately form an enzyme-acetoacetyl-CoA complex upon aerobic addition to butyryl-CoA dehydrogenase purified from Megasphaera elsdenii, implying the presence of crotonase activity. This behaviour remains even after treatment with 6M urea, which destroys the activity of the main crotonase fraction from M. elsdenii. Flavin-sensitised photoinactivation destroys residual crotonase and dehydrogenase activities in parallel. Butyryl-CoA dehydrogenase thus has intrinsic crotonase activity with a turnover rate (0.05 min-1) about 0.02% of the figure for dehydrogenase activity. Mechanistic implications are discussed.

Acyl Coenzyme A↗
PubMed1992

The essential active-site lysines of clostridial glutamate dehydrogenase. A study with pyridoxal-5'-phosphate.

Glutamate dehydrogenase (GDH) of Clostridium symbiosum, like GDH from other species, is inactivated by pyridoxal 5'-phosphate (pyridoxal-P). This inactivation follows a similar pattern to that for beef liver GDH, in which a non-covalent GDH-pyridoxal-P complex reacts slowly to form a covalent complex in which pyridoxal-P is in a Schiff's-base linkage to lysine residues. [formula: see text] The equilibrium constant of this first-order reaction on the enzyme surface determines the final extent of inactivation observed [S. S. Chen and P. C. Engel (1975) Biochem. J. 147, 351-358]. For clostridial GDH, the maximal inactivation obtained was about 70%, reached after 10 min with 7 mM pyridoxal-P at pH 7. In keeping with the model, (a) inactivation became irreversible after reduction with NaBH4. (b) The NaBH4-reduced enzyme showed a new absorption peak at 325 nm. (c) Km values for NAD+ and glutamate were unaltered, although Vmax values were decreased by 70%. Kinetic analysis of the inactivation gave values of 0.81 +/- 0.34 min-1 for k3 and 3.61 +/- 0.95 mM for k2/k1. The linear plot of 1/(1-R) against 1/[pyridoxal-P], where R is the limiting residual activity reached in an inactivation reaction, gave a slightly higher value for k2/k1 of 4.8 +/- 0.47 mM and k4 of 0.16 +/- 0.01 min-1. NADH, NAD+, 2-oxoglutarate, glutarate and succinate separately gave partial protection against inactivation, the biggest effect being that of 40 mM succinate (68% activity compared with 33% in the control). Paired combinations of glutarate or 2-oxoglutarate and NAD+ gave slightly better protection than the separate components, but the most effective combination was 40 mM 2-oxoglutarate with 1 mM NADH (85% activity at equilibrium). 70% inactivated enzyme showed an incorporation of 0.7 mM pyridoxal-P/mol subunit, estimated spectrophotometrically after NaBH4 reduction, in keeping with the 1:1 stoichiometry for the inactivation. In a sample protected with 2-oxoglutarate and NADH, however, incorporation was 0.45 mol/mol, as against 0.15 mol/mol expected (85% active). Tryptic peptides of the enzyme, modified with and without protection, were purified by HPLC. Two major peaks containing phosphopyridoxyllysine were unique to the unprotected enzyme. These peaks yielded three peptide sequences clearly homologous to sequences of other GDH species. In each case, a gap at which no obvious phenylthiohydantoin-amino-acid was detected, matched a conserved lysine position. The gap was taken to indicate phosphopyridoxyllysine which had prevented tryptic cleavage.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1992

The glutamate dehydrogenase gene of Clostridium symbiosum. Cloning by polymerase chain reaction, sequence analysis and over-expression in Escherichia coli.

The gene encoding the NAD(+)-dependent glutamate dehydrogenase (GDH) of Clostridium symbiosum was cloned using the polymerase chain reaction (PCR) because it could not be recovered by standard techniques. The nucleotide sequence of the gdh gene was determined and it was overexpressed from the controllable tac promoter in Escherichia coli so that active clostridial GDH represented 20% of total cell protein. The recombinant plasmid complemented the nutritional lesion of an E. coli glutamate auxotroph. There was a marked difference between the nucleotide compositions of the coding region (G + C = 52%) and the flanking sequences (G + C = 30% and 37%). The structural gene encoded a polypeptide of 450 amino acid residues and relative molecular mass (M(r) 49,295 which corresponds to a single subunit of the hexameric enzyme. The DNA-derived amino acid sequence was consistent with a partial sequence from tryptic and cyanogen bromide peptides of the clostridial enzyme. The N-terminal amino acid sequence matched that of the purified protein, indicating that the initiating methionine is removed post-translationally, as in the natural host. The amino acid sequence is similar to those of other bacterial GDHs although it has a Gly-Xaa-Gly-Xaa-Xaa-Ala motif in the NAD(+)-binding domain, which is more typical of the NADP(+)-dependent enzymes. The sequence data now permit a detailed interpretation of the X-ray crystallographic structure of the enzyme and the cloning and expression of the clostridial gene will facilitate site-directed mutagenesis.

Amino Acid Sequence↗
PubMed1992

Subunit assembly and active site location in the structure of glutamate dehydrogenase.

The three-dimensional crystal structure of the NAD(+)-linked glutamate dehydrogenase from Clostridium symbiosum has been solved to 1.96 A resolution by a combination of isomorphous replacement and molecular averaging and refined to a conventional crystallographic R factor of 0.227. Each subunit in this multimeric enzyme is organised into two domains separated by a deep cleft. One domain directs the self-assembly of the molecule into a hexameric oligomer with 32 symmetry. The other domain is structurally similar to the classical dinucleotide binding fold but with the direction of one of the strands reversed. Difference Fourier analysis on the binary complex of the enzyme with NAD+ shows that the dinucleotide is bound in an extended conformation with the nicotinamide moiety deep in the cleft between the two domains. Hydrogen bonds between the carboxyamide group of the nicotinamide ring and the side chains of T209 and N240, residues conserved in all hexameric GDH sequences, provide a positive selection for the syn conformer of this ring. This results in a molecular arrangement in which the A face of the nicotinamide ring is buried against the enzyme surface and the B face is exposed, adjacent to a striking cluster of conserved residues including K89, K113, and K125. Modeling studies, correlated with chemical modification data, have implicated this region as the glutamate/2-oxoglutarate binding site and provide an explanation at the molecular level for the B type stereospecificity of the hydride transfer of GDH during the catalytic cycle.

Amino Acid Sequence↗
PubMed1991

Functional studies of a glutamate dehydrogenase with known three-dimensional structure: steady-state kinetics of the forward and reverse reactions catalysed by the NAD(+)-dependent glutamate dehydrogenase of Clostridium symbiosum.

Steady-state kinetic properties of glutamate dehydrogenase from Clostridium symbiosum are reported. Rates with NADP(H) are over three hundred times lower than with NAD(H) under identical conditions. The 3-acetyl pyridine and 6-deamino adenine analogues of NAD+, on the other hand, are used almost as well as NAD+ itself. Amino acid specificity is very tight at both pH 7 and pH 9. The best alternative substrate of those tested, L-alpha-amino-gamma-nitraminobutyrate, gave only 0.5% of the rate seen with glutamate. With 400 microM NAD+ a 160-fold variation of the glutamate concentration gave a linear Eadie plot apart from slight inhibition at the highest concentrations. With 40 mM L-glutamate and varied [NAD+], the Eadie plot appeared linear between 1.6 microM and 60 microM and again between 60 microM and 2000 microM, but the slopes of the two lines differed by a factor of 8.4. This striking pattern is not attributable to impurities in the coenzyme or to changes in the state of aggregation of the enzyme. For the high concentration range (greater than 60 microM NAD+), the presence of all four linear terms in the reciprocal form of the initial rate equation indicates a sequential mechanism. Similar measurements made for APAD+ and dnNAD+ show no sign of non-linearity in the Eadie plot over the wide concentration ranges explored. In the reductive amination direction, with NADH as coenzyme, linear reciprocal plots were obtained for all three substrates. Systematic variation of concentrations led via primary, secondary and tertiary plots to all eight possible initial-rate parameters in a linear reciprocal initial-rate equation. Compulsory-order and enzyme-substitution mechanisms appear to be excluded, and a random route to the central complex seems the only possibility compatible with the results.

Amines↗
PubMed1991

The partial amino acid sequence of the NAD(+)-dependent glutamate dehydrogenase of Clostridium symbiosum: implications for the evolution and structural basis of coenzyme specificity.

The amino acid sequence is reported for CNBr and tryptic peptide fragments of the NAD(+)-dependent glutamate dehydrogenase of Clostridium symbiosum. Together with the N-terminal sequence, these make up about 75% of the total sequence. The sequence shows extensive similarity with that of the NADP(+)-dependent glutamate dehydrogenase of Escherichia coli (52% identical residues out of the 332 compared) allowing confident placing of the peptide fragments within the overall sequence. This demonstrated sequence similarity with the E. coli enzyme, despite different coenzyme specificity, is much greater than the similarity (31% identities) between the GDH's of C. symbiosum and Peptostreptococcus asaccharolyticus, both NAD(+)-linked. The evolutionary implications are discussed. In the 'fingerprint' region of the nucleotide binding fold the sequence Gly X Gly X X Ala is found, rather than Gly X Gly X X Gly. The sequence found here has previously been associated with NADP+ specificity and its finding in a strictly NAD(+)-dependent enzyme requires closer examination of the function of this structural motif.

Amino Acid Sequence↗
PubMed1991

Specific diagnosis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency in dried blood spots by a polymerase chain reaction (PCR) assay detecting a point-mutation (G985) in the MCAD gene.

The discovery of a point-mutation, adenine-to-guanine, at position 985 in the gene coding for MCAD (G985), gave the basis for an easy and specific polymerase chain reaction test. We tested the specificity of such a PCR based assay and detected correctly G985 and A985 in sequence verified cDNA clones. We showed that the G985 mutation is present in genomic DNA from 48 of 50 patients with confirmed MCAD deficiency, originating from various European countries, Australia and the USA. On the basis of this high frequency of the G985 mutation among patients, we improved and optimized the assay with respect to reliability and convenience for routine diagnostic and screening purposes. As little as 2 microliters blood from filter-paper blood-spots (Guthrie spots) is sufficient for the test.

Acyl-CoA Dehydrogenase↗
PubMed1991

The most common mutation causing medium-chain acyl-CoA dehydrogenase deficiency is strongly associated with a particular haplotype in the region of the gene.

RFLP haplotypes in the region containing the medium-chain acyl-CoA dehydrogenase (MCAD) gene on chromosome 1 have been determined in patients with MCAD deficiency. The RFLPs were detected after digestion of patient DNA with the enzymes BanII. PstI and TaqI and with an MCAD cDNA-clone as a probe. Of 32 disease-causing alleles studied, 31 possessed the previously published A----G point-mutation at position 985 of the cDNA. This mutation has been shown to result in inactivity of the MCAD enzyme. In at least 30 of the 31 alleles carrying this G985 mutation a specific RFLP haplotype was present. In contrast, the same haplotype was present in only 23% of normal alleles (P less than or equal to 3.4 x 10(-18)). These findings are consistent with the existence of a pronounced founder effect, possibly combined with biological and/or sampling selection.

Acyl-CoA Dehydrogenase↗