Studies on the anaplerotic mechanisms of micro-organisms.

Mutants of Escherichia coli were isolated and identified as lacking key enzymes of specific anaplerotic equences. With these muntants it was shown that growth on pyruvate or precursors thereof necessitates the functioning of an acetyl-COA dependant PEP carboxylase, and that growth on acetate requires the operation of isocitrate lyase. The growth of all the PSP carboxylase-less mutants on acetate was inhibited by addition of pyaruvate or precursors thereof. Analysis of this effect of pyruvate and substances derived from it on the behaviour of whole cells and cell-free extracts suggested that the phenomenan is due to the inhibition by PEP of isocitrate lyase. Further studies with mutants which lacked the ability to convert pyruvate to PEP or which were derepressed for isocitrate lyase suggested that the inhibition of isocitrate lyase by PEP was of physiological significance and provides a means whereby the rate of operation of the glyoxylate cycle is controlled. Isocitrato lyase has been partially purified and its properties investigated. An anaplerotic enzyme of glyoxylate metabolism, tartronic semi-aldehyde/(TSA) reductase was shown to be formed during growth of E. coli on DL-glycerate. Studies on the behaviour of mutants devoid of glycollic oxidase or PEP carboxylase or citrate synthase activities indicate, however, that the formation of TSA reductase is not an indication of a pathway of glycerate metabolism distinct from the glycolytic sequences: rather it is concluded that it functions as one component of the enzymic system catalysing the interconversion of the D and L isomers of glycerate.