Mechanistic and Stereochemical Studies on the Glycine Reductase of Clostridium sticklandii

Abstract

Clostridial glycine reductase multienzyme complex which catalyses the reaction :Glycine + ADP + Pi + 2H -+ Acetate + ATP + NH3 was solubilised and fractionated essentially according to the method of Stadtman [T. C. Stadtman (1970) Methods Enzymol. I7A, 956 -9661 into two components : protein A and 'glycine reductase' fraction. A reconstituted system obtained by combining the two components in the presence of dithiothreitol catalysed the conversion of glycine into acetate concomitant with the phosphorylation of ADP to ATP. Using the reconstitued system, in which the unwanted enzymic activity catalyzing an exchange of the ct hydrogen atoms of glycine with the protons of the medium had been greatly reduced, it was found that the conversion of (2RS)-[2-14C, 2-3H1]glycine (3H/'4C = 7.16) into acetate (3H/14C = 7.03) was attended by the retention of both the C-2 hydrogen atoms of glycine. Conversion of (2S)-[2-2H1, 2-3Hl]glycine and (2R)-[2-2H1, 2-3Hl]glycine by the reconstituted system gave (2S)-acetate and (2R)-acetate respectively showing that the reductive deamination of glycine occurs through an inversion of configuration. The cumulative information available on the glycine reductase reaction is embodied in a hypothetical mechanism of action for the enzyme.

Identification of acetylphosphate as the product of clostridial glycine reductase: evidence for an acyl enzyme intermediate

“…One equivalent of ATP is synthesized from 1 equiv of orthophosphate and 1 equiv of ADP concomitant with the reduction of glycine (Tanaka & Stadtman, 1979). However, in the absence of ADP the amount of acetate formed is one-third that formed in the presence of ADP (Stadtman et al, 1958;Barnard & Akhtar, 1979).…”

Selenium-dependent Growth and Glycine Fermentation by Clostridium purinolyticum

“…Cell-free extracts of C. sticklandii showed a somewhat lower activity of the enzyme although the value was about 10-fold higher than those reported by Stadtman & Elliott (1956). This might be due to the more gentle storage conditions established by Barnard & Akhtar (1979) and also used by us. The activities determined for glycine decarboxylase, formyltetrahydrofolate synthetase, methenyltetrahydrofolate cyclohydrolase and methylenetetrahydrofolate dehydrogenase were of about the same order as known for purine-fermenting clostridia (Champion & Rabinowitz, 1977).…”

“…Cells were always harvested at the end of the exponential growth phase and stored anaerobically at -20 "C. They were ruptured by French press treatment, and cell-free extracts were prepared as described by Leonhardt & Barnard & Akhtar (1979), glycine decarboxylase (EC 2 . 1 .…”

Purine and glycine metabolism by purinolytic clostridia

“…C. cylindrosporum contained 0.03 U/ mg of protein; no activity could be detected in extracts of C. acidiurici by the standard test (36). The enzyme activity is known to be labile (4,16,36), and this may account for failure to find the activity in C. acidiurici.…”