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

II. EFFECT OF ACETYLATION ON MOLECULAR PROPERTIES

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

The concentration-dependent reversible association-dissociation reaction of beef liver glutamate dehydrogenase is found to be affected by treatment with acetic anhydride. Under different conditions, acetylation in Tris-acetate buffer yields either a fully active or inactive species which can no longer undergo association. As determined by light-scattering and equilibrium sedimentation, the molecular weight of the acetylated enzyme, either active or inactive, is 400,000 ± 25,000, the same as observed for native enzyme when reversibly dissociated. In this paper, association of the enzyme is considered as a monomer-polymer process with monomer being defined as enzyme of molecular weight 400,000, even though the monomer itself consists of eight peptide chains of molecular weight 50,000.

Acetylation of partially associated native enzyme at low concentrations of acetic anhydride decreases enzymatic activity by 80% with little change in the weight average molecular weight. At higher acetic anhydride concentrations, the molecular weight drops sharply to that of the monomer, and the activity is completely lost.

It is concluded that different functional groups are responsible for catalytic activity and for monomer-tetramer association. Further, as measured by ninhydrin, only one amino group per peptide chain of molecular weight 50,000 is responsible for the changes in catalytic activity and regulatory properties, and one to two different amino groups per peptide chain are involved in the monomer-tetramer association. The reactivity of these latter groups, but not the former, is influenced by the state of association of the protein.

The mode of association of the enzyme is different when adenosine diphosphate plus reduced diphosphopyridine nucleotide are present since a pentamer (containing 40 peptide chains) rather than a tetramer (with 32 peptide chains) is formed at high enzyme concentrations. From the effect of acetylation on activity and molecular weight in the presence and absence of ADP and DPNH, it is concluded that pentamer, but not tetramer, formation involves the same functional group as that involved in over-all catalytic activity and regulatory properties.

The results obtained concerning the kinetic and molecular properties are discussed in terms of a minimum of four different types of interactions. These are intrapeptide chain, interpeptide chain, monomer-monomer interactions in the absence of, and monomer-monomer interactions in the presence of, ADP and DPNH.

Tyrosyl and sulfhydryl groups are not involved in the changes which are observed upon treatment with acetic anhydride.

The advantage of performing acetylation experiments in Tris-acetate buffer is discussed.

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