Studies of glutamate dehydrogenase: analysis of functional areas and functional groups.
1. It is shown by limited tryptic digestion of beef liver glutamate dehydrogenase under native conditions that the amino terminus of the polypeptide chain is located at the surface of the molecule. End-group analysis after trypsin treatment yields aspartic acid as the new N-terminal amino acid while the C-terminal threonine remains unchanged. 2. NADH, especially in the presence of 2-oxoglutarate, protects the enzyme against tryptic degradation. In the absence of the coenzyme, glutamate dehydrogenase is rapidly inactivated. 3. The regulatory effects of ADP and GTP are only slightly altered by trypsin. A small shift of the pH dependence of the activation by ADP is observed. 4. The quaternary structure of the unimer of the enzyme is not affected by limited tryptic digestion indicating that the N-terminal part of the polypeptide chain is not located in the contact domains between the polypeptide  chains. The association of the hexamer to large associated particles is reduced but not abolished. 5. It is shown by treatment of the enzyme with iodo[2(-14)C]acetic acid as well as with Ellman's reagent that the six - SH groups of the polypeptide chain are buried and not accessible to these reagents in phosphate buffer. In Tris buffer they become exposed and react in the order 89, 55, 197, 115, 270, 319. This together with the result that in Tris buffer the rat of inactivation caused by trypsin is higher than in phosphate buffer indicates that Tris buffer changes drastically the properties of the enzyme. 6. Cross-linking of the enzyme molecule with bifunctional reagents and subsequent dodecylsulfate-polyacrylamide electrophoresis shows that the six identical polypeptide chains are arranged in two groups of three. 7. The implications of these results for the tertiary and quaternary structure of beef liver glutamate dehydrogenase are discussed.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007094:Imides; D007656:Ketoglutaric Acids; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D009243:NAD; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011487:Protein Conformation; D014357:Trypsin
D007094:Imides; D007656:Ketoglutaric Acids; D046911:Macromolecular Substances; D010446:Peptide Fragments; D009243:NAD; D005969:Glutamate Dehydrogenase; D014357:Trypsin