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On the Role of Amino Groups in the Structure and Function of Glutamate Dehydrogenase

I. EFFECT OF ACETYLATION ON CATALYTIC AND REGULATORY PROPERTIES

From the ¹ From the Department of Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri 63110

Acetic anhydride has been used to modify particular amino acid residues of beef liver glutamate dehydrogenase. Acetylation in tris(hydroxymethyl)aminomethane acetate buffer at pH 7.15 or 8 results in a striking increase in both the guanosine triphosphate and zinc dissociation constants, a decrease in the adenosine diphosphate dissociation constant, normal kinetics at high reduced diphosphopyridine nucleotide levels, and a loss of adenosine triphosphate inhibition. On the other hand, Michaelis constants for coenzymes and other substrates are unaffected by such treatment. Also unaffected are the binding constants for purine nucleotides in the absence of coenzyme.

It is concluded that the kinetic changes observed on acetylation reflect a loss of the interaction between the coenzyme-active site and the purine nucleotide-binding site.

Acetylation also results in loss of enzymatic activity. However, it is shown that substances which inhibit the catalytic reaction protect the enzyme against both the loss of activity and loss of interaction, provided that the acetylation is carried out in the presence of coenzyme in addition to the inhibitory compound. It is suggested that there is a relationship between the functional group acetylated and a rate-limiting step in the enzymatic reaction on the basis of this type of protection and, furthermore, that this functional group is responsible for the interaction between the coenzyme and purine nucleotide site in the active enzyme. It is postulated that this functional group does not lie within any particular nucleotide site, but is masked in the presence of inhibitory substances presumably by a conformational change in the enzyme.

It is also concluded, on the basis of the different effects of purine nucleotides on activity and their differing ability to protect against acetylation in the presence of DPNH, that the ADP and GTP sites do not completely overlap.

Finally, two distinct types of interactions, one interpeptide chain and the other intrapeptide chain, are postulated to explain the kinetic properties of native and acetylated enzyme.

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