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P C Engel

Publications and source records attributed to P C Engel.

At least 37 records · Page 2Linked to original sources
PubMed1998

Protein thermostability in extremophiles.

Thermostability of a protein is a property which cannot be attributed to the presence of a particular amino acid or to a post synthetic modification. Thermostability seems to be a property acquired by a protein through many small structural modifications obtained with the exchange of some amino acids and the modulation of the canonical forces found in all proteins such as electrostatic (hydrogen bonds and ion-pairs) and hydrophobic interactions. Proteins produced by thermo and hyperthermophilic microorganisms, growing between 45 and 110 degrees C are in general more resistant to thermal and chemical denaturation than their mesophilic counterparts. The observed structural resistance may reflect a restriction on the flexibility of these proteins, which, while allowing them to be functionally competent at elevated temperatures, renders them unusually rigid at mesophilic temperatures (10-45 degrees C). The increased rigidity at mesophilic temperatures may find a structural determinant in increased compactness. In thermophilic proteins a number of amino acids are often exchanged. These exchanges with some strategic placement of proline in beta-turns give rise to a stabilization of the protein. Mutagenesis experiments have confirmed this statement. From the comparative analysis of the X-ray structures available for several families of proteins, including at least one thermophilic structure in each case, it appears that thermal stabilization is accompanied by an increase in hydrogen bonds and salt bridges. Thermostability appears also related to a better packing within buried regions. Despite these generalisations, no universal rules can be found in these proteins to achieve thermostability.

Adenylate Kinase↗
PubMed1998

Group specific antibodies against the putative AMP-binding domain signature SGTTGXPKG in peptide synthetases and related enzymes.

The superfamily of adenylate forming enzymes including peptide synthetases, acyl-CoA synthetases and insect luciferases is readily identified by the signature sequence SGTTGXPKG. This sequence including an invariant lysyl residue is located in a disordered loop region and was predicted to be of significant antigenicity. Antibodies were generated employing YTSGTTGRPKGC attached to bovine serum albumin and have been successfully used to identify respective enzymes and adenylate forming domains in multienzyme systems. These include the delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetases of Aspergillus nidulans and Acremonium chrysogenum, gramicidin S synthetase 1 and tyrocidine synthetase 1 from Bacillus brevis, acetyl-CoA synthetase from Alcaligenes eutrophus and a putative peptide synthetase from Metarhizium anisopliae. Weaker or no reactions are observed when the amino acid in position X in the protein is non-basic or hydrophobic, which is respectively the case for gramicidin S synthetase 1 and luciferase.

Adenosine Monophosphate↗
PubMed1998

Use of protein engineering to explore subunit interactions in an allosteric enzyme: construction of inter-subunit hybrids in Clostridium symbiosum glutamate dehydrogenase.

Hybrids of different forms of clostridial glutamate dehydrogenase (GDH) have been constructed in order to probe the basis of allosteric interaction in this hexameric enzyme. It was shown that the C320S mutant, which is fully active and shows allosteric behaviour similar to that of the wild-type enzyme, can also be renatured after unfolding in urea. Mixtures of unfolded wild-type and C320S subunits gave rise to hybrids upon refolding. A purely random reassembly would lead to a simple binomial distribution. However there was a slightly better overall recovery of wild-type subunits and there appears to be a tendency for rapidly formed structured wild-type subunits in a mixture to nucleate further refolding in a way that biases the final distribution against the formation of C320S hexamers. Only the wild-type subunits in such hybrid mixtures are able to react with Ellman's reagent, 5,5'-dithiobis-(2-nitrobenzoate) (DTNB). Accordingly, after modification of hybrid hexamers with DTNB only the mutant subunits can bind NAD+. This permits fractionation on an NAD+-agarose affinity column. The elution pattern in itself indicates cooperativity since DTNB modification of just one subunit in a 1:5 wild-type/C320S hybrid largely abolished binding to the column. Kinetic studies were carried out on a fractionated preparation in which hexamers containing only one C320S subunit and five wild-type subunits were the predominant active species. Measurements of activity were made both before and after treatment with an excess of beta-mercaptoethanol to remove the blocking thionitrobenzoate moieties. Before beta-mercaptoethanol treatment this sample, with only one active subunit per hexamer, gave strictly hyperbolic (Michaelis-Menten) kinetics with NAD+ at pH 7.0, whereas after beta-mercaptoethanol (all six subunits now active) the markedly kinked Eadie-Hofstee plot characteristic of wild-type enzyme was obtained. On the other hand the sigmoid response to glutamate at high pH persisted (Hill coefficient=3.6) even without beta-mercaptoethanol, reflecting the fact that the inactive subunits can still bind glutamate. Beta-mercaptoethanol treatment restored full positive cooperativity (Hill coefficient=5.2). These results prove beyond doubt that the non-classical kinetic behaviour of clostridial GDH is a direct result of interaction between NAD+ binding sites on the six (normally) identical subunits of a hexamer.

Allosteric Regulation↗
PubMed1997

Determinants of substrate specificity in the superfamily of amino acid dehydrogenases.

The subunit of the enzyme glutamate dehydrogenase comprises two domains separated by a cleft harboring the active site. One domain is responsible for dinucleotide binding and the other carries the majority of residues which bind the substrate. During the catalytic cycle a large movement between the two domains occurs, closing the cleft and bringing the C4 of the nicotinamide ring and the Calpha of the substrate into the correct positioning for hydride transfer. In the active site, two residues, K89 and S380, make interactions with the gamma-carboxyl group of the glutamate substrate. In leucine dehydrogenase, an enzyme belonging to the same superfamily, the equivalent residues are L40 and V294, which create a more hydrophobic specificity pocket and provide an explanation for their differential substrate specificity. In an attempt to change the substrate specificity of glutamate dehydrogenase toward that of leucine dehydrogenase, a double mutant, K89L,S380V, of glutamate dehydrogenase has been constructed. Far from having a high specificity for leucine, this mutant appears to be devoid of any catalytic activity over a wide range of substrates tested. Determination of the three-dimensional structure of the mutant enzyme has shown that the loss of function is related to a disordering of residues linking the enzyme's two domains, probably arising from a steric clash between the valine side chain, introduced at position 380 in the mutant, and a conserved threonine residue, T193. In leucine dehydrogenase the steric clash between the equivalent valine and threonine side chains (V294, T134) does not occur owing to shifts of the main chain to which these side chains are attached. Thus, the differential substrate specificity seen in the amino acid dehydrogenase superfamily arises from both the introduction of simple point mutations and the fine tuning of the active site pocket defined by small but significant main chain rearrangements.

Amino Acid Oxidoreductases↗
PubMed1997

Intersubunit communication in hybrid hexamers of K89L/A163G/S380A and C320S mutants of glutamate dehydrogenase from Clostridium symbiosum.

The triple mutant K89L/A163G/S380A (inactive with glutamate but active with L-Nle and L-Met) and C320S (fully active with glutamate, entirely inactive with L-Nle and L-Met, and also lacking reactive cysteine) mutant of glutamate dehydrogenase (EC 1.4.1.2) of Clostridium symbiosum could be completely denatured by urea with the loss of structure and activity. The mutants denatured by urea could be reassociated to give stable hexamers with recovery of activity of approximately 67% by dilution in 0.1 M potassium phosphate buffer (pH 7.0) containing 2 mM NAD+. The native, urea-denatured, and renatured states of mutant enzymes were characterized by size exclusion chromatography on FPLC and native PAGE. Intersubunit hybrid hexamers containing five subunits of triple mutant and one subunit of C320S mutant were constructed by in vitro subunit hybridization followed by affinity chromatography. Kinetic analysis showed that a 5:1 hybrid hexamer, with only one C320S subunit able to bind NAD+ after DTNB modification, shows classical Michaelis-Menten kinetics with regard to NAD+. This contrasts with the apparent negative co-operativity shown by pure C320S hexamers and suggests that the interaction in NAD+ binding among subunits is eliminated in the hybrid. After removal of thionitrobenzoate, however, all of the subunits in the hybrid are able to bind NAD+. In this state the hybrid enzyme showed slight deviation from classical behavior with regard to NAD+, indicating reintroduction of some level of allosteric interaction. The hybrid hexamer also showed much reduced co-operativity with glutamate at pH 8.8, with a Hill coefficient of 3 for DTNB-treated hybrid (as compared to 5.2 for the pure C320S mutant) and 2.2 for the untreated hybrid. The fact that co-operativity in glutamate binding is not entirely eliminated correlates with evidence that the triple mutant subunits, though inactive toward glutamate, can nevertheless still bind this amino acid.

Alanine↗
PubMed1997

Re-activation of Clostridium symbiosum glutamate dehydrogenase from subunits denatured by urea.

In a study of the re-activation of urea-denatured clostridial glutamate dehydrogenase (GDH) the maximum re-activation achieved without any added ligands was about 6%, but with NAD+ and 2-oxoglutarate in combination about 70%. NAD+ alone was also effective but 2-oxoglutarate was not, in striking contrast with the opposite pattern for protection of this enzyme against unfolding in urea [Aghajanian, Martin and Engel (1995) Biochem. J. 311,905-910]. The extent of re-activation was not increased by raising the incubation temperature to 37 degrees C and was independent of the time of enzyme denaturation. CD and fluorimetric studies showed that dilution of denatured enzyme into potassium phosphate buffer led to rapid (half-time <3-5 s)formation of 'structured' intermediates with secondary structure similar to that of native enzyme. These intermediate molecules were inactive, behaved as monomers on a size-exclusion column, and were unable to associate to give the native hexameric structure. Addition of NAD+ facilitated isomerization of these 'structured' monomers into a form(s) capable of re-activation. A side effect in the refolding process was non-specific aggregation, depending on final enzyme concentration. The hexamer fraction from re-activated samples, however, showed the same specific activity as native enzyme. The portion of the enzyme that is not lost through aggregation thus appears to regain the native structure fully. Detailed time-course studies showed that re-activation follows second-order kinetics, suggesting that formation of a dimer may be the rate-limiting step. The possible mechanism for the unfolding and refolding processes of clostridial GDH and effects of coenzyme and substrate on these are discussed in relation to the known crystal structure.

Clostridium↗
PubMed1997

Kinetic studies on the binding of 1,N6-etheno-NAD+ to glutamate dehydrogenase from Clostridium symbiosum.

The mechanism of the binding of reduced coenzyme (NAD+) to clostridial glutamate dehydrogenase (GDH) was determined by transient kinetics. The fluorescent 1,N6-ethenoadenine analogue of NAD+ (epsilonNAD+) was used as a probe of nucleotide binary and ternary complex formation because the binding of NAD+ is optically silent. The kinetics of epsilonNAD+ binding were consistent with a 3-step binding process. The enzyme was found to oscillate between two conformational forms, termed E1 and E2, in the presence and absence of L-glutamate. However, L-glutamate shifted the equilibrium from 96.8% to 99% of the enzyme in the E1 form. The rapid-equilibrium binding of epsilonNAD+ to the E2 form was rate limited by a slow isomerisation of the ternary complex as the binary complex became saturated with epsilonNAD+. The L-glutamate binary complex had a greater affinity for the coenzyme (Kd = 11 microM) than the free enzyme (Km = 39 microM), indicative of a positive interaction of the substrate and coenzyme binding sites. Steady-state studies were also indicative of a positive interaction in the formation of the catalytic complex, with this complex having a Kd for epsilonNAD+ of 6.8 microM. Consequently, there is stabilization of successive complexes on the reaction pathway.

Clostridium↗
PubMed1997

Alteration of the quaternary structure of glutamate dehydrogenase from Clostridium symbiosum by a single mutation distant from the subunit interfaces.

X-ray crystallographic studies have previously shown that glutamate dehydrogenase from Clostridium symbiosum is a homohexamer. Mutation of the active-site aspartate-165 to histidine causes an alteration in the structural properties of the enzyme. The mutant enzyme, D165H exists predominantly as a single species of lower molecular mass than the wild-type enzyme as indicated by gel filtration and sedimentation velocity analysis. The latter technique gives an S20,w value for D165H of (6.07 +/- 0.01)S which compares with (11.08 +/- 0.01)S for the wild-type, indicative of alteration of the homohexameric quaternary structure of the native enzyme to a dimeric form, a result confirmed by sedimentation equilibrium experiments. Further support for this is provided by chemical modification by Ellman's reagent of cysteine-144 in the mutant, a residue which is buried at the dimer-dimer interface in the wild-type enzyme and is normally inaccessible to modification. The results suggest a possible structural route for communication between the active sites and subunit interfaces which may be important for relaying signals between subunits in allosteric regulation of the enzyme.

Chromatography, Gel↗
PubMed1996

Construction of a dimeric form of glutamate dehydrogenase from Clostridium symbiosum by site-directed mutagenesis.

By using site-directed mutagenesis, Phe-187, one of the amino-acid residues involved in hydrophobic interaction between the three identical dimers comprising the hexamer of Clostridium symbiosum glutamate dehydrogenase (GDH), has been replaced by an aspartic acid residue. Over-expression in Escherichia coli led to production of large amounts of a soluble protein which, though devoid of GDH activity, showed the expected subunit M(r) on SDS-PAGE, and cross-reacted with an anti-GDH antibody preparation in Western blots. The antibody was used to monitor purification of the inactive protein. F187D GDH showed altered mobility on non-denaturing electrophoresis, consistent with changed size and/or surface charge. Gel filtration on a calibrated column indicated an M(r) of 87000 +/- 3000. The mutant enzyme did not bind to the dye column routinely used in preparing wild-type GDH. Nevertheless suspicions of major misfolding were allayed by the results of chemical modification studies: as with wild-type GDH, NAD+ completely protected one-SH group against modification by DTNB, implying normal coenzyme binding. A significant difference, however, is that in the mutant enzyme both cysteine groups were modified by DTNB, rather than C320 only. The CD spectrum in the far-UV region indicated no major change in secondary structure in the mutant protein. The near-UV CD spectrum, however, was less intense and showed a pronounced Phe contribution, possibly reflecting the changed environment of Phe-199, which would be buried in the hexamer. Sedimentation velocity experiments gave corrected coefficients S20,W of 11.08 S and 5.29 S for the wild-type and mutant proteins. Sedimentation equilibrium gave weight average molar masses M(r,app) of 280000 +/- 5000 g/mol. consistent with the hexameric structure for the wild-type protein and 135000 +/- 3000 g/mol for F187D. The value for the mutant is intermediate between the values expected for a dimer (98000) and a trimer (147000). To investigate the basis of this, sedimentation equilibrium experiments were performed over a range of protein concentrations. M(r,app) showed a linear dependence on concentration and a value of 108 118 g/mol at infinite dilution. This indicates a rapid equilibrium between dimeric and hexameric forms of the mutant protein with an equilibrium constant of 0.13 l/g. An independent analysis of the radial absorption scans with Microcal Origin software indicated a threefold association constant of 0.11 l/g. Introduction of the F187D mutation thus appears to have been successful in producing a dimeric GDH species. Since this protein is inactive it is possible that activity requires subunit interaction around the 3-fold symmetry axis. On the other hand this mutation may disrupt the structure in a way that cannot be extrapolated to other dimers. This issue can only be resolved by making alternative dimeric mutants.

Aspartic Acid↗