The final two enzymes in the CoA biosynthetic pathway, phosphopantetheine adenylyltransferase (PPAT; EC 2.7.7.3) and dephospho-CoA kinase (DPCK; EC 2.7.1.24), are separate proteins in prokaryotes, but exist as a bifunctional enzyme in pig liver. In the present study we have obtained sequence information from purified pig-liver enzyme, and identified the corresponding cDNA in a number of species. The human gene localizes to chromosome 17q12-21 and contains regions with sequence similarity to the monofunctional Escherichia coli DPCK and PPAT. The recombinant 564-amino-acid human protein confirmed the associated transferase and kinase activities, and gave similar kinetic properties to the wild-type pig enzyme.

Urea-induced effects in clostridial glutamate dehydrogenase (GDH, EC 1.4.1.2) were studied by spectrophotometry, circular dichroism, FPLC, affinity chromatography and PAGE. Denaturation of enzyme occurred over a narrow range of urea concentrations (2.5-3.5 M), accompanied by inactivation of enzyme with a similar rate constant. The contribution of instantaneous inhibition by urea was also ascertained. FPLC studies of urea-treated GDH gave no evidence for dissociated oligomeric fragments of the hexamer in the presence of subdenaturing concentrations of urea. Likewise a mixture of fully 5,5'-dithiobis-(2-nitrobenzoic acid)-modified GDH hexamers and unmodified enzyme in 2 M urea failed to give rise to hybrid molecules. Exposure of unmodified GDH to high concentrations of urea led to the dissociation of hexamers to denatured monomers followed by association to form non-specific high-M(r) aggregates. This conclusion was confirmed by native gradient PAGE experiments. Various specific ligands stabilized the enzyme against urea-induced inactivation, succinate and 2-oxoglutarate being particularly effective. This protection of the native state was enhanced in ternary complexes, and the complex most resistant to urea-induced inactivation was the productive ternary complex GDH-NADH-2-oxoglutarate. Native gradient PAGE experiments indicate that these protecting ligands preserve the native hexameric structure of GDH.

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.

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][906][907][908][909][910]. The extent of re-activation was not increased by raising the incubation temperature to 37 mC 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,

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.