Lactic dehydrogenase and cytochrome b2 of baker's yeast. Enzymic and chemical properties of the crystalline enzyme

Abstract

Chemical and enzymic studies with crystalline yeast lactic dehydrogenase are described. The absorption spectrum of the lactate-reduced enzyme shows maxima at 556.5, 528, 424, 330 and 265 m[mu]; [epsilon] at 556.5 m[mu] is 38.8 X 103. There is 1 mole each of protohaem and of riboflavin phosphate present as prosthetic groups/80 000 g of enzyme. No significant amount of non-haem iron, copper, cobalt, molybdenum or other related metal is present. There is 1 g atom of mg/80,000 g of enzyme; this is probably associated with the deoxyribopolynucleotide component of the crystalline material. Oxidation by hydrogen peroxide, and by added Cu2+ ions under aerobic conditions, causes dissociation of the flavin prosthetic group and loss of dehydrogenase activity. Ethylenediaminetetraacetate protects against metal-catalyzed inactivation. The mechanism of the inactivation process is discussed. The term cytochrome b2 applies to the native, enzymically active flavohaemoprotein. A haemoprotein product containing no riboflavin phosphate and devoid of enzymic activity may be obtained from cytochrome b2. The enzyme catalyses the oxidation of glycollate, L-lactate and [alpha]-hydroxybutyrate only. Lactate is oxidized most rapidly. The activation energies for reduction of heart-muscle cytochrome c, potassium ferricyanide and methylene blue under specific conditions are 7[center dot]2, 7[center dot]6 and 8[center dot]7 kcal./mole, respectively. Under specified conditions, reduction of heart-muscle cytochrome c proceeds by a 1st-order reaction, and of ferricyanide by a zero-order reaction. The enzymic and chemical properties of the crystalline cytochrome b2 are compared with those of non-crystalline preparations obtained by other workers.