Functional Requirements of the Active Site Position 185 in the Human Enzyme Galactose-1-phosphate Uridylyltransferase

B Booth Quimby,Lance Wells,Keith D Wilkinson,Judith L Fridovich-Keil

doi:10.1074/jbc.271.43.26835

Abstract

The active site of galactose-1-phosphate uridylyltransferase (GALT) includes a HPH sequence that has been conserved in all species examined from Escherichia coli to humans. The crystal structure of the E. coli enzyme suggests that this proline is important in positioning the active site histidine (His-166) near the substrate. To examine the role of this proline in the homologous human sequence, we have performed saturating mutagenesis at Pro-185 within human GALT and characterized each resultant mutant enzyme using a yeast expression system. Activity analyses in crude lysates indicated that only proline at position 185 produced wild-type levels of activity, although five other amino acids, Ala, Gly, Ser, Gln, and Glu, all produced partially active enzymes. Western blot analyses of the GALT proteins in these lysates demonstrated that abundance varied from 9-118% of wild-type and was independent of activity. All five active mutant proteins were purified and characterized with regard to specific activity, apparent Km for both substrates, and temperature-dependence of activity. Finally, modeling of these mutations onto the conserved E. coli active site structure was performed. Together, these results provide functional evidence demonstrating the critical role of Pro-185 in facilitating the transferase reaction.

Highlights

Summary and Significance—In summary, position 185 of human GALT is important for the rate of reaction of the enzyme with the first substrate, UDPG, but less so for the reaction of the uridyl enzyme with the second substrate, Gal-1-P
Substituting amino acids can be clustered into five groups: Pro; Ser and Ala; Gly; Glu and Gln; and all others
No other mutants show detectable enzymatic activity, the abundance of these proteins varies from ϳ10 to Ͼ100% of control. These results are fully consistent with the accepted catalytic model for this enzyme and are consistent with the published x-ray structure of the E. coli enzyme

Summary

Introduction

Summary and Significance—In summary, position 185 of human GALT is important for the rate of reaction of the enzyme with the first substrate, UDPG, but less so for the reaction of the uridyl enzyme with the second substrate, Gal-1-P. Substituting amino acids can be clustered into five groups: Pro (wild type); Ser and Ala; Gly; Glu and Gln; and all others. P185S and P185A are approximately 15-fold slower than wildtype at forming uridyl enzyme, have a temperature optimum of about 37 °C, and approximately 30 –50% wild-type abundance.

Results

Conclusion