The importance of conserved residues in human liver UDPglucose pyrophosphorylase.
Comparison of the amino acid sequences of five eukaryotic UDPglucose pyrophosphorylases has identified a number of conserved residues that may be important for substrate binding or catalysis. Using the cloned cDNA for the human liver enzyme, we have investigated the role of several of these residues by site-directed mutagenesis. Changing the single conserved cysteine (residue 123) to serine resulted in an active enzyme, as did mutating the single concerned histidine (residue 266) to arginine. The two conserved tryptophans were each altered to serine; W218S is active while W333S is not. In the latter case, the enzyme does not appear to fold correctly, and a similar result was obtained by mutation to lysine at one (residue 391) of the four conserved arginines. The other three arginines are not essential, as judged by the observation that R389H, R422Q and R445H are all active. The kinetic properties of each active mutant were investigated and in most cases were found to be similar to those of wild-type. The most dramatic change is a sevenfold increase in the Km for magnesium pyrophosphate with C123S. Overall, none of these conserved residues appears to be essential for activity, although such a role cannot be ruled out for W333 and R391 where mutation resulted in defective folding.
Chang
H Y
HY
Department of Molecular and Cellular Biology, Chang-Gung College of Medicine and Technology, Kwei-San, Taiwan, Republic of China.
Peng
H L
HL
Chao
Y C
YC
Duggleby
R G
RG
D000818:Animals; D002417:Cattle; D017931:DNA Primers / Q000737:chemistry; D004023:Dictyostelium / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000737:chemistry* / Q000378:metabolism
D017931:DNA Primers; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase