Malolactic enzyme in Lactobacillus murinus.

Abstract

The malolactic enzyme of Lactobacillus murinus is inducible. The induction is produced by L-malic acid only in the presence of glucose and amino acids and occurs at the transcription level. The enzyme, purified to homogeneity, has a Mr of 220,000 and consists of 2 apparently identical subunits (Mr = 110,000) that were observed after treatment with sodium dodecyl sulphate. NAD+ protected the enzyme against inactivation and its addition, after dissociation, restored the malolactic activity. Maximum enzyme activity was observed at 37 degrees C and pH 5.5. At pH values substantially different from the optimum, a positive cooperativity between substrate molecules was observed. The activation energy of the reaction was 8,000 and 16,200 cal mol-1 for temperatures above and below 30 degrees C, respectively. Malolactic enzyme catalyzes the NAD+ and manganese-dependent reaction; L-malate----L-lactate + CO2. The stoichiometry of the reaction was confirmed. The malolactic transformation occurs by a compulsory-order mechanism. NAD+ bound first to the protein, independently of malate concentration. Mn2+ acts as an allosteric activator. Malate bound to the complex enzyme-NAD-Mn2+. Oxamate, fructose 1,6-diphosphate and malonate acted as non-competitive inhibitors, whereas citrate and L-tartrate produced a competitive inhibition. This enzyme can be distinguished from the malic enzyme of pigeon liver (E.C.1.1.1.40) and from the true malic enzymes (E.C.1.1.1.38 and E.C.1.1.1.39).