THE IMPORTANCE OF CONSERVED RESIDUES IN HUMAN LIVER UDPGLUCOSE PYROPHOSPHORYLASE

Comparison of the amino acid sequences of five eukaryotic UDPglucose p yrophosphorylases has identified a number of conserved residues that m ay be important for substrate binding or catalysis. Using the cloned c DNA for the human liver enzyme, we have investigated the role of sever al of these residues by site-directed mutagenesis. Changing the single conserved cysteine (residue 123) to serine resulted in an active enzy me, as did mutating the single conserved histidine (residue 266) to ar ,ginine. The two conserved tryptophans were each altered to serine, W2 18S 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 mut ation to lysine at one (residue 391) of the four conserved arginines. The other three arginines are not essential, as judged by the observat ion that R3S9H, 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 sev enfold increase in the K-m for magnesium pyrophosphate with C123S. Ove rall, none of these conserved residues appears to be essential for act ivity, although such a role cannot be ruled out for W333 and R391 wher e mutation resulted in defective folding.