Studies of Glutamate Dehydrogenase

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Abstract
Cross-linking of the unimer of glutamate dehydrogenase from beef liver (consisting of 6 polypeptide chains each having a MW of 56,000) with dimethyladipimidate and subsequent analysis by sodium dodecylsulfate electrophoresis shows predominantly the trimeric species (MW 168,000). Treatment with dimethylimidates of other chain length yields significantly less trimeric species indicating that the amino groups being cross-linked are within a distance of about 0.85 nm. Comparison of the molar amount of incorporated [14C]dimethyladipimidate with the number of modified amino groups (determined with trinitrobenzenesulfonic acid) shows that although 8-9 of the 34 amino groups have reacted, only 2-3 of them are involved in cross-links. Reaction with dimethylimidates inactivates the enzyme. The loss of the activity is partly concomitant to cross-linking to the trimeric species and not simply due to the modification of essential lysine residues. This is supported by the fact that, although more lysine residues react with monofunctional methylimidates, the loss of activity is reduced. Purified chymotryptic and tryptic peptides of the radioactive-labeled trimeric species were subjected to sequence analysis. Six peptides containing 75% of the total label were identified: 1 involves the amino-terminal residue Ala-1 and the others involve Lys-105, Lys-154, Lys-269, Lys-358 and Lys-399. Quantitative analysis of the specific radioactivity of each peptide/mol Lys leads to the conclusion that only Lys-105, Lys-154, Lys-269 and Lys-358 participate in cross-links, Lys-269 and Lys-358, respectively, being at isologous and Lys-105 cross-linked with Lys-154 at heterologous contact domains of the enzyme. A model for the planar arrangement of the trimeric species in the quaternary structure of glutamate dehydrogenase is discussed. It includes isologous and heterologous contact areas between the polypeptide chains.