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Further investigation of the amino-acid sequence of bovine glutamate dehydrogenase (EC 1.4

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Pyridoxal inactivates glutamate dehydrogenase presumably by forming an imine with the €-amino group of lysine-97, as in the case of pyridoxal 5'-phosphate. The equilibrium constants for imine formation a t varying pH values (Kprr) have been calculated from the initial concentrations of enzyme and pyridoxal and the final degree of inactivation. The variation of KDw with pH has been related to the dissociation constants of the reactive €-amino group, pyridoxal, and the product imine, and a single equilibrium constant for imine formation. When this treatment was applied to the inactivation of glutamate dehydrogenase by pyridoxal G lutamate dehydrogenase (L-glutamate: DPN(TPN) oxidoreductase (deaminating), EC 1.4.1.3) occupies an important position in the nitrogen metabolism of mammals since it catalyzes a reaction which is the major pathway for the interconversion of a-amino group nitrogen and ammonia (Meister, 1965 ; Frieden, 1963a). Although other important enzymes of amino acid metabolism generally employ pyridoxal 5'-phosphate as a cofactor in transamination or decarboxylation reactions, glutamate dehydrogenase is inhibited by this compound through the formation of an imine with the €-amino group of a lysyl residue (Anderson e f a/., 1966). In addition, this enzyme is subject to allosteric regulation by a variety of nucleoside polyphosphates (Frieden, 1963a,b); for example, GTP acts as an inhibitor and ADP as an acti-

The hydrolyses of -carboxyphenyl /3-D-glucopyranoside (o-CP-Gl) and o-carboxyphenyl 2-acetamido-2-deoxy-/3-D-glucopyranoside (o-CP-NAG) have been studied at 78.2°( µ = 0.3) between pH 0.75 and 11.80. The pH-log kobsd profiles for these compounds are characterized by a plateau rate in the acid region, followed by a descending leg of approximate slope of -1.0 above approximately pH 4. The plateau and descending leg are found to have kinetically equivalent interpretations as intramolecular carboxyl group participation in the hydrolysis, or specific acid catalyzed hydrolysis of the carboxylate anion form of the glycosides. The former interpretation has been shown to be correct on the basis that the log &rat« values for the calculated constants for specific acid catalyzed hydrolysis of the dissociated glycosides exhibit significant positive deviations from Hammett ( ) plots constructed from the rate constants for specific acid catalysis of the hydrolysis of glucosides not containing carboxyl groups. In addition, o-CP-NAG is found to have a plateau rate (kc) significantly greater than o-CP-Gl. To account for this result, intramolecular 2-acetamido group participation as well as intramolecular carboxyl group participation is deduced. A mechanism for the hydrolysis of o-CP-NAG involving concerted intramolecular carboxyl group general acid and intramolecular acetamido group nucleophilic catalysis is proposed. By analogy, the spontaneous hydrolysis of o-CP-Gl is concluded to occur by intramolecular carboxyl group general acid catalysis. A previous study of the intramolecular acetamido group catalyzed hydrolysis of substituted phenyl 2-acetamido-2-deoxy-3-D-glucopyranosides is extended to show that the rate of this reaction is dependent upon aglycone leaving group tendencies (i.e., the Hammett p~v alue is found to be +2.6). The activation parameters for the spontaneous hydrolyses of o-CP-Gl, o-CP-NAG, and o-nitrophenyl 2-acetamido-2-deoxy-3-D-glucopyranoside are determined. A value of -TAS* for the hydrolysis of o-CP-NAG which is 2 kcal/mol larger than -TAS* for the hydrolysis of o-CP-Gl is attributed to the necessity of properly orienting an additional catalytic group, the acetamido group, in the rate-determining transition state. The spontaneous rate of hydrolysis (kc) of the model o-CP-NAG is found to be of similar magnitude to k^t for the enzyme-catalyzed hydrolysis of a similar glycoside under comparable conditions. The relationship of these results to the mechanism of lysozyme is discussed.

Abstract. A tentative but almost complete amino acid sequence for the subunit peptide chain of bovine liver glutamate dehydrogenase indicates a minimal size of 506 residues with a molecular weight of 56,100, in accord with the physical size of the subunit of 55,900. Inactivation with pyridoxal 5'-phosphate, followed by reduction with sodium borohydride, has permitted identification of the essential lysine as residue 97. Nitration of tyrosine-412 is accompanied by loss of the allosteric inhibitory effect of guanosine triphosphate.Comparison of the sequences of glutamate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase has indicated that only two 12-residue sequences are similar in the two enzymes; this sequence includes reactive lvsine-97 of the former enzyme.Glutamate dehydrogenase (EC 1.4.1.3) occupies a central position in mammalian nitrogen metabolism since the reaction which it catalyzes provides the major pathway for the interconversion of a-amino group nitrogen and ammonia. Other important enzymes of amino acid metabolism usually employ pyridoxal 5'-phosphate in transamination or decarboxylation as the initial reaction step. The dehydrogenase is also of interest because its activity is subject to allosteric regulation by a variety of nucleoside polyphosphates, e.g., GTP is a strong inhibitory effector and ADP an activator.1 We have undertaken a study of the structure of glutamate dehydrogenase2 for a variety of reasons: first, because little is yet known regarding the structures and active sites of dehydrogenases; second, because this enzyme could serve as a useful model in attempts to understand the regulatory processes of multichain enzymes in general and of dehydrogenases in particular; third, because it would be useful to ascertain possible evolutionary relationships among the many dehydrogenases that utilize the pyridine nucleotide coenzymes, DPN and TPN. At present, only the sequence of glyceraldehyde-3-phosphate dehydrogenase (EC 1.2