The Tentative Identification in Escherichia coli of a Multienzyme Complex with Glycolytic Activity

Abstract

Penicillin spheroplasts of E. coli were ruptured osmotically, by freezing and thawing, or mechanically. Differential centrifugation sedimented 20-30% of the glycolytic enzymes without increasing their specific activities. There was evidence of distinct groups of sedimenting enzymes; growth on different C sources could influence the distribution. Sucrose gradient studies gave no evidence of enzyme association but provided estimations of the MW of each enzyme which were close to those subsequently observed on gel filtration. Using the determined MW and a literature value for specific activity, the measured activity ratio of the enzymes was compared with that expected from an equimolar mixture. All values agreed within a factor of 5, except for hexokinase. The relative roles of hexokinase and phosphotransferase in E. coli are briefly considered. An equimolar multienzyme aggregate of all the enzymes of glycolysis would have a MW of about 1.6 .times. 106. Chromatography on a Biogel column yielded 1 fraction, corresponding to MW of 1.6 .times. 106, which contained a proportion of all the glycolytic enzyme studied; the remaining portion of each enzyme activity was eluted from the column at the position expected from its individual MW. The fraction of MW 1,600,000 was tested for complete glycolysis pathway activity and found not to be different from a reconcentrated mixture of the separated enzymes. Both the eluted and the reconstructued systems showed unexpected activity changes at different protein concentrations. The specific radioactivity of pyruvate formed by these systems from [14C]glucose 6-phosphate was reduced by the presence of unlabeled 3-phosphoglycerate, but by less than would have been expected had the latter been able to participate fully in glycolytic activity. These reparations were apparently capable of selectivity compartmenting glycolytic intermediates. EM investigation of both systems showed large numbers of regular 30 nm diameter particles which, on disruption, appeared to be composed of smaller units; it is possible that these particles may have been aggregates containing glycolytic enzymes. The possible advantages of a glycolytic multienzyme complex are briefly discussed.