Maltose transacetylase of Escherichia coli. Mapping and cloning of its structural, gene, mac, and characterization of the enzyme as a dimer of identical polypeptides with a molecular weight of 20,000.
malQ mutants, lacking amylomaltase, cannot grown on maltose. However, when maltose is present in the medium, it can be accumulated to high internal levels. In a subsequent slow reaction, accumulated maltose becomes acetylated and leaks back into the medium. The enzyme responsible for this acetylation uses acetyl-CoA as acetyl donor and can be measured in crude extracts (Boos, W., Ferenci, T., and Shuman, H. A. (1981) J. Bacteriol. 146, 725-732). The structural gene for the enzyme, which we named mac, was mapped at 10.4 min on the Escherichia coli linkage map. We cloned a 3.4-kilobase pair PstI-EcoRI DNA fragment containing the mac gene. Cell-free extracts of a strain harboring the multicopy plasmid were used to purify the maltose-transacetylating activity to apparent homogeneity. On sodium dodecyl sulfate-polyacrylamide gels the enzyme exhibited a molecular weight of 20,000. Using molecular sieve chromatography, a molecular weight of 40,000 was determined for the native enzyme. Therefore, the enzyme is a dimer of two identical subunits. At a sugar concentration of 100 mM the enzyme acetylates glucose, maltose, mannose, galactose, and fructose in decreasing relative rate of 1, 0.55, 0.20, 0.07, 0.04. Maltotriose and other oligosaccharides were acetylated with 2% of the rate determined for glucose. The Km for glucose and maltose were 62 and 90 mM, and the Vmax was 0.20 and 0.11 mmol/min x mg enzyme, respectively.
Brand
B
B
Department of Biology, University of Konstanz, Federal Republic of Germany.
Boos
W
W
D000123:Acetyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003065:Coenzyme A / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D008040:Genetic Linkage; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009154:Mutation; D015183:Restriction Mapping
D004269:DNA, Bacterial; D046911:Macromolecular Substances; D000123:Acetyltransferases; C033939:maltose acetyltransferase; D003065:Coenzyme A