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

Abstract

Penicillin spheroplasts of Escherichia 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, however, evidence of distinct groups of sedimenting enzymes; growth on different carbon sources could influence the distribution. Sucrose gradient studies gave no evidence of enzyme association but provided estimations of the molecular weight of each enzyme which were close to those subsequently observed on gel filtration. Using the determined molecular weight 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 five, 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 molecular weight of about 1.6 × 106. Chromatography on a Biogel column yielded one fraction, corresponding to a molecular weight of 1.6 × 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 molecular weight. The fraction of mol. wt 1600 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 reconstructed 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 unlabelled 3‐phosphoglycerate, but by less than would have been expected had the latter been able to participate fully in glycolytic activity. This result indicates that these preparations were capable of selectivity compartmenting glycolytic intermediates. Electron microscope 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.

Interactions between glycolytic enzymes and components of the cytomatrix.

“…trapping, non-specific binding, binding due to cell lysis, and reassociation in non-physiological buffer systems). Nevertheless, in the case ofthe individual glycolytic enzymes, a considerable quantity ofsupportive evidence ofthe reality ofbinding of these enzymes to structure in cells and tissues has been provided by a variety oftechniques (12,13,15,19), and this body of evidence now seems quite compelling. In particular, it has been demonstrated that most of the glycolytic enzymes have a particular affinity for actin and actin-containing structures, such as the thin filaments of muscle and the microfilaments of the cytoskeleton (3,12,14), and substantial associations between such structures and individual glycolytic enzymes have been described for both in vivo and in vitro conditions .…”

Production and Tolerance of Ethanol in Relation to Phospholipid Fatty-acyl Composition in Saccharomyces cerevisiae NCYC 431

“…cerevisiae synthesizes two or more isomers of several of the enzymes that catalyse reactions on the Embden-Meyerhof-Parnas pathway (Fraenkel,198 1). Moreover, several reports indicate that the enzymes may be membranebound (Rothstein et al, 1959;Green et al, 1965;Atkinson, 1969;Sols & Marco, 1970), although it has yet to be demonstrated that they exist in a multi-enzyme complex as in Escherichia coli (Mowbray & Moses, 1976).…”

A role of RnlA in the RNase LS activity from Escherichia coli

“…First, it stoichiometrically binds to RnlA with a high affinity. Second, TpiA is known to exist in a large complex containing proteins involved in carbohydrate metabolism (Mowbray and Moses, 1976). These facts may imply that some of the other 11 proteins associate with RnlA via TpiA.…”