Specific activation of a tyrosine----glycine mutant of delta 5-3-ketosteroid isomerase by phenols.
A key unknown still to be explored concerning the mechanism of delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni is the extent of the proton transfer between tyrosine-14 of the enzyme and the C-3 carbonyl oxygen of the steroid substrate. This report is a preliminary study of a system we are developing to allow us eventually to use a Brønsted analysis to measure this transfer. We describe the construction of an expression vector and tyrosine-14----glycine-14 mutant of the enzyme and its specific activation, in the manner of chemical rescue, by a variety of phenolic compounds. We suggest that the binding region of phenol is very tight and that the level of activation may be a result of steric constraints as well as of differences in the pKa' of the phenol.
Brooks
B
B
Department of Biological Chemistry, School of Medicine, University of California, Davis 95616.
Benisek
W F
WF
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005822:Genetic Vectors; D005998:Glycine; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010636:Phenols / Q000494:pharmacology*; D010957:Plasmids; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D013251:Steroid Isomerases / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine
D010636:Phenols; D011994:Recombinant Proteins; D014443:Tyrosine; D013251:Steroid Isomerases; C022660:steroid delta-isomerase; D005998:Glycine