The metabolism of C2 compounds in micro-organisms. 7. Preparation and properties of crystalline tartronic semialdehyde reductase

Abstract

Tartronic semialdehyde reductase was purified 200-fold and crystallized from glycollate-grown Pseudomonas ovalis Chester. The enzyme catalyzed the reduction of tartronic semialdehyde to glyceric acid with DPNH or TPNH as electron donor. Km for tartronic semialdehyde at pH 8.5 was measured to be 2 x 10-4[image]. Km for DPNH under these conditions was 2 x 10-5[image] and for TPNH 5 x 10-5[image]. The crystalline enzyme oxidized 160 [mu]moles of DPNH/min./mg. of protein at pH 8.5, 23[degree], in the presence of tartronic semialdehyde. On the basis of the measured weight-average molecular weight MW of 91,000, this gave a turnover number of 14,600 moles of DPNH oxidized/min./mole of enzyme. The enzyme was shown also to catalyze the oxidation of glycerate with concomitant reduction of DPN. The equilibrium constant K at 23[degree] was 2 x 10-6 at pH 7.5 and 1.6 x 10-5 at pH 8.5; Kh was calculated to be 5.1 x 10-14[image]. From these data, [DELTA]G[image] at pH 7, 25[degree], was determined as 8.6 kcal./mole and AG[degree] as 18 kcal./mole. Similarly, the oxidation-reduction potential for the reaction [image] was found to be Eo = -0.092v at 25[degree] and pH 7. The enzymic oxidation of glycerate to tartronic semialdehyde was also demonstrated by coupling it to the reduction of pyruvate to lactate, in the presence of DPN and crystalline lactic dehydrogenase, and to the reduction of 2,6-dichlorophenol-indophenol in the presence of DPN and DPNH dehydrogenase. The latter system served as a convenient assay of the enzyme. The enzyme catalyzed the reduction only of C3 compounds of structure CHO.R.CO2H, where R = CH.OH, CH2 or CO. Tartronic semialdehyde (R = CH.OH) reacted more than five times as fast as any other substrate. The enzyme catalyzed the oxidation only of glyceric acid and of hydroxypyruvate. The significance of these findings to the pathways of biosynthesis from glyoxylate is discussed.