The Action Pattern of Amylomaltase from Escherichia coli

Abstract

Amylomaltase, the inducible 4-.alpha.-glucanotransferase of E. coli strain ML, was purified to homogeneity. Its specific activity with a commercial maltose substrate was 500 mkat[millikatal (= 1 mmol/s)]/kg protein (30 .mu.mol glucose formed min-1 mg protein-1). The purified enzyme, dependent on buffer concentration, exists in interconvertible low molecular-weight (apparent MW 71,000) and high-molecular-weight (apparent MW 370,000) forms. The specificity of amylomaltase was redefined. The enzyme was formerly thought to be a glucosyltransferase, catalyzing the transfer of single glucosyl units, and maltose was regarded as its most important substrate. Amylomaltase is now shown to exhibit both glucosyl-transfer and 4-.alpha.-glucanosyl-transfer specificity. 4-.alpha.-Glucanosyl chains containing up to at least 9 glucosyl units can be transferred. The transfer reaction by which amylomaltase action was originally expressed apparently does not take place, i.e., maltose + maltose .dblarw. maltotriose + glucose and maltose has a restricted role as a substrate. This may be due to the inability of maltose to function as a donor substrate, serving only as an acceptor substrate. When a maltodextrin serves as a donor, that portion of the molecule transfered by the enzyme is that containing the nonreducing-end-group. Enzyme action on chromatographically pure maltose is characterized by a lag phase in the time course of glucose release. The lag phase is overcome by addition of priming (catalytic) concentrations of maltotriose or higher maltodextrins. An autocatalytic reaction mechanism involving the generation of primer molecules is proposed to explain the action of the enzyme on maltose. The redefined action pattern of amylomaltase is consistent with the redefined role of the enzyme in the utilization of exogenous and endogenous, 1,4-.alpha.-glucans by E. coli.