Alpha-isopropylmalate synthase from yeast: purification, kinetic studies, and effect of ligands on stability.
alpha-Isopropylmalate synthase, the first specific enzyme in leucine biosynthesis, was purified approximately 100-fold from extracts of Saccharomyces sp. (strain 60615), the most effective step being specific elution with the feedback inhibitor leucine from a hydroxyapatite column. In the early steps of purification, special care was taken to protect the synthase against proteolytic activities. The apparent molecular weight of the enzyme as determined from gel filtration on a calibrated column was 137,000 in the absence and 121,000 in the presence of leucine. Inhibition by leucine was specific and strongly pH-dependent, with the leucine concentration necessary for half-maximal inhibition increasing about 10-fold as the pH increased from 7.5 to 8.5. Within this pH range, catalytic activity remained almost unchanged. The apparent K(m) values for the two substrates were found to be 16 mum for alpha-ketoisovalerate and 9 mum for acetyl-coenzyme A. K(+) was required for full activity, the apparent K(a) value being 2 mm. Leucine inhibition was of the mixed type, resulting in decreased V(max) and increased apparent K(m) values forboth substrates. Whereas no cooperative effects were observed with either substrate, positive cooperativity was seen with leucine in the presence of saturating substrate concentrations. Leucine and, to a lesser extent, alpha-ketoisovalerate stabilized the purified enzyme against heat-inactivation. The presence of acetyl-coenzyme A, on the other hand, accelerated the inactivation. In subsequent experiments, coenzyme A was recognized as the actual inactivating ligand, being effective even at lower temperatures and in concentrations which were estimated to be in the range of the enzyme concentration.
D000217:Acyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D002458:Cell Fractionation; D011232:Chemical Precipitation; D002845:Chromatography; D002850:Chromatography, Gel; D003065:Coenzyme A / Q000494:pharmacology; D003998:Dicarboxylic Acids; D004789:Enzyme Activation; D005246:Feedback; D006018:Glycols; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006882:Hydroxyapatites; D007651:Keto Acids / Q000494:pharmacology; D007700:Kinetics; D007930:Leucine / Q000494:pharmacology; D008970:Molecular Weight; D012440:Saccharomyces / Q000201:enzymology* / Q000254:growth & development; D013307:Streptomycin; D013431:Sulfates; D014631:Valerates
D003998:Dicarboxylic Acids; D006018:Glycols; D006882:Hydroxyapatites; D007651:Keto Acids; D013431:Sulfates; D014631:Valerates; D000217:Acyltransferases; D007930:Leucine; D003065:Coenzyme A; D013307:Streptomycin