ACETYLTRANSFER PRECEDES URIDYLYLTRANSFER IN THE FORMATION OF UDP-N-ACETYLGLUCOSAMINE IN SEPARABLE ACTIVE-SITES OF THE BIFUNCTIONAL GLMU PROTEIN OF ESCHERICHIA-COLI

The GlmU protein is a bifunctional enzyme with both acetyltransferase and uridylyltransferase (pyrophosphorylase) activities which catalyzes the transformation of glucosamine-1-P, UTP, and acetyl-CoA to UDP-N-a cetylglucosamine [Mengin-Lecreulx, D., & van Heijenoort, J. (1994) J. Bacteriol. 176, 5788-5795], a fundamental precursor in bacterial pepti doglycan biosynthesis and the source of activated N-acetylglucosamine in lipopolysaccharide biosynthesis in Gram-negative bacteria. In the w ork described here, the GlmU protein and truncation variants of GlmU ( N- and C-terminal) were purified and kinetically characterized for sub strate specificity and reaction order. It was determined that the GLmU protein first catalyzed acetyltransfer followed by uridylyltransfer, The N-terminal portion of the enzyme was capable of only uridylyltrans fer, and the C-terminus catalyzed only acetyltransfer. GlmU demonstrat ed a 12-fold kinetic preference (k(cat)/K-m, 3.1 x 10(5) versus 2.5 x 10(4) L . mol(-1). s(-1)) for acetyltransfer from acetyl-CoA to glucos amine-1-P as compared to UDP-glucosamine. No detectable uridylyltransf er from UTP to glucosamine-1-P was observed in the presence of GlmU; h owever, the enzyme was competent in catalyzing the formation of UDP-N- acetylglucosamine from UTP and N-acetylglucosamine-1-P (k(cat)/K-m 1.2 x 10(6) L . mol(-1). s(-1)). A two active site model for the GlmU pro tein was indicated both by domain dissection experiments and by assay of the bifunctional reaction. Kinetic studies demonstrated that a pre- steady-state lag in the production of UDP-N-acetylglucosamine from ace tyl-CoA, UTP, and glucosamine-1-P was due to the release and accumulat ion of steady-state levels of the intermediate N-acetylglucosamine-1-P .