Selective proteolysis of cytosolic aspartate aminotransferase by a new microbial protease.
A protease from Streptomyces violaceochromogenes (Murao, S., Nishino, Y., & Maeda, Y. (1984) Agric. Biol. Chem. 48, 2163-2166) is known to inactivate pig heart aspartate aminotransferase [EC 2.6.1.1]. Chemical analysis of the core proteins and peptide fragments produced upon proteolysis of the aminotransferase revealed that peptide bond cleavage occurred specifically at Leu 20 with concomitant inactivation. Neither inactivation nor peptide bond cleavage was observed with the mitochondrial isoenzyme. The proteolytically produced derivative 21-412 of the cytosolic isoenzyme retained approximately 0.1% enzymic activity for transamination with natural dicarboxylic substrates. The pyridoxal form of the derivative 21-412 was fully converted by cysteinesulfinate or alanine to the pyridoxamine form and conversely the pyridoxamine form of the derivative was also fully converted by 2-oxoglutarate or pyruvate into the pyridoxal form, indicating that the derivative was still catalytically competent. However, the rates of reaction with dicarboxylic substrates were much reduced whereas the rates with monocarboxylic substrates remained at an order of magnitude similar to that observed with the native enzyme. Thus the NH2-terminal segment appears to be an import structural component which determines the substrate specificity of aspartate aminotransferase for dicarboxylic keto and amino acids. A substantial alteration in the molecular structure accompanying the loss of the NH2-terminal 20 residues was also reflected by the decrease in heat stability and in the lowering of the pKa value for His 68, which is involved in the intersubunit interaction of this dimeric enzyme.
D000818:Animals; D001219:Aspartate Aminotransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification / Q000494:pharmacology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D008970:Molecular Weight; D009206:Myocardium / Q000166:cytology / Q000201:enzymology; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013552:Swine; D013997:Time Factors
D007527:Isoenzymes; D001219:Aspartate Aminotransferases; D010450:Endopeptidases