A microtiter plate assay for UDP-galactopyranose mutase, an essential cell wall biosynthetic enzyme of Mycobacterium tuberculosis, was developed. The assay is based on the release of tritiated formaldehyde from UDP-galactofuranose but not UDP-galactopyranose by periodate and was used to identify a uridine-based enzyme inhibitor from a chemical library.The enzyme UDP-galactopyranose mutase (Glf), which catalyzes the formation of UDP-galactofuranose (UDP-galf) from UDP-galactopyranose (UDP-galp), plays a key role in the biosynthesis of the galactofuran component of the cell wall of Mycobacterium tuberculosis, as shown in Fig. 1. Many attributes of Glf suggest it as a drug target. Glf has been shown to be essential for mycobacterial growth (5). No analogous enzymatic reaction takes place in humans. UDP-galactopyranose mutase from Escherichia coli has been crystallized, its structure has been determined (3, 6), and the structure of the protein from M. tuberculosis has been just been determined to 2.5Å (Jim Naismith, personal communication). However, a convenient assay appropriate for inhibitor searches has been lacking.Previously, Glf has been assayed via high-performance liquid chromatographic separation of UDP-galf and UDP-galp (1). In an earlier attempt to provide a simpler assay, UDP-galf was used as the substrate (in the reverse direction) and the UDPgalp product was converted with two helper enzymes to UDPglucuronic acid with concomitant reduction of NAD to NADH. However, this assay is not suitable for screening for inhibitors of the enzyme because of the difficulty of obtaining large amounts of UDP-galf, concerns about looking for inhibitors when running the reaction in the reverse direction, and the drawback that helper enzymes are required. Thus, we designed a microtiter plate-based assay to screen for potential inhibitors of this enzyme.The M. tuberculosis Glf enzyme was expressed in E. coli (8). Purification of Glf from the E. coli extract resulted in an enzyme preparation that was less reliable in activity; this is believed to be due to irreversible loss of the flavin adenine dinucleotide cofactor (4). The E. coli strain (BL21) used to express M. tuberculosis Glf is a galE mutant and also does not have the E. coli glf gene found in E. coli K-12 (2, 7). Consequently, E. coli proteins do not act on the UDP-Galp substrate. In the assay described below, the amount of enzyme was adjusted so that about 5% of the UDP-galp present was converted to UDP-galf. This was a compromise in that enough radioactivity was produced for reproducibility but equilibrium had not been reached at 7% UDP-Galf (4).The theory behind the new assay for Glf is presented in Fig. 2. Thus, tritiated formaldehyde is released from the product (UDP-[6-3 H]Galf) but not the substrate (UDP-[6-3 H]Galp) after treatment with periodate. Therefore, to assay the uridinebased library (9), 23 l of a cocktail consisting of 25 mM HEPES (pH 7.2), 25 nmol of MgCl 2 , 25 nmol of freshly prepared NADH, and 2.5 nmol (ϳ0.08 Ci) of UDP-[6-3 H]galactopyranose...