Galactose mutarotase: purification, characterization, and investigations of two important histidine residues.
Galactose mutarotase catalyzes the interconversion of alpha- and beta-anomers of aldoses and is a recently identified member of the gal operon of Escherichia coli and participant in the Leloir pathway [Bouffard et al. (1994) J. Mol. Biol. 244, 269-278]. We report the purification and characterization of this enzyme, as well as mechanistic studies involving chemical modification with diethylpyrocarbonate (DEPC) and site-directed mutagenesis demonstrating the significance of two conserved histidine residues. The enzyme lacks metal ions and oxidoreduction cofactors, and an extinction coefficient of (6.2 +/- 0.4) x 10(4) M-1 cm-1 has been measured by quantitative amino acid analysis. The catalytic mechanism is likely concerted general acid/general base. Experiments involving modification with DEPC suggest that a histidine is essential and is protected by substrate. Furthermore, site-directed mutagenesis of two conserved histidines was performed, and characterization of these mutants (His104Gln and His175Asn) illustrates the significance of these residues. Kinetic analysis of H104Q demonstrates an increase in KM of about 600-fold, a decrease in kcat of approximately 7-fold, and a 4000-fold decrease in kcat/KM as compared to the wild-type enzyme. The activity of His175Asn mutant, on the other hand, was too low to be measured accurately, and His 175 remains a candidate for the general base. These mutants were also subjected to DEPC modification, and results are consistent with the presence of two important histidines positioned closely together in the active site.
Institute for Enzyme Research, University of Wisconsin-Madison 53705, USA.
J A
JA
Beebe
P A
PA
Frey
D017124:Conserved Sequence / Q000235:genetics; D004047:Diethyl Pyrocarbonate; D004926:Escherichia coli / Q000201:enzymology; D005690:Galactose / Q000737:chemistry; D006639:Histidine / Q000737:chemistry* / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D008670:Metals / Q000032:analysis; D016297:Mutagenesis, Site-Directed; D014534:UDPglucose 4-Epimerase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*
D008670:Metals; D006639:Histidine; D014534:UDPglucose 4-Epimerase; C020551:galactose epimerase; D004047:Diethyl Pyrocarbonate; D005690:Galactose