ACTIVE-SITE TOPOLOGY OF BOVINE CHOLESTEROL SIDE-CHAIN CLEAVAGE CYTOCHROME-P450 (P450SCC) AND EVIDENCE FOR INTERACTION OF TYROSINE-94 WITH THE SIDE-CHAIN OF CHOLESTEROL
Ia. Pikuleva et al., ACTIVE-SITE TOPOLOGY OF BOVINE CHOLESTEROL SIDE-CHAIN CLEAVAGE CYTOCHROME-P450 (P450SCC) AND EVIDENCE FOR INTERACTION OF TYROSINE-94 WITH THE SIDE-CHAIN OF CHOLESTEROL, Archives of biochemistry and biophysics, 322(1), 1995, pp. 189-197
Combining site-directed mutagenesis with analysis of the active-site t
opology of bovine cholesterol sidechain cleavage cytochrome P450scc (P
450scc), we have investigated the roles of tyrosine residues 93 and 94
on substrate binding. Four single mutants (Y93A, Y93S, Y94A, and Y94S
) and one double mutant (Y93S/Y94S) were examined. The largest increas
e in K-s was observed for binding of cholesterol and 25-hydroxycholest
erol to the Y94S mutant (similar to 5.5-fold), with a smaller increase
(<2.5-fold) for binding of 22-hydroxycholesterol. Mutation of Y94 thu
s appears to influence the interaction with cholesterol, 25-hydroxycho
lesterol, and possibly 22-hydroxycholesterol. Y93 is not involved in b
inding of 22- and 25-hydroxycholesterol but may interact with choleste
rol. The active-site topologies of P450scc and its mutants were probed
by reaction with three arylhydrazines. The N-arylprotoporphyrin IX re
gioisomer patterns obtained with phenyl- and 2-naphthylhydrazine indic
ate that the active site is primarily open above pyrrole ring A and su
ggest that a region some distance above pyrrole ring D is also open. T
he single mutations Y93S, Y93A, Y94A, and Y94S do not detectably alter
the regioisomer patterns obtained with the phenyl- and 2-naphthyl pro
bes, but a small, reproducible change is observed with the 2-naphthyl
probe for the Y93S/Y94S double mutant, The conformational alteration i
mplied by this change could not be detected by titration with 22- and
25-hydroxycholesterol but is detectable by titration with cholesterol.
The results indicate that cholesterol binds over pyrrole ring D of th
e heme in bovine P450scc, strongly suggest that Y94 interacts with the
side chain of cholesterol, and provide evidence that the side chains
of 22- and 25-hydroxycholesterol bind to a different region of the act
ive site than the side chain of cholesterol. (C) 1995 Academic Press I
nc.