SUBSTRATE DOCKING ALGORITHMS AND PREDICTION OF THE SUBSTRATE-SPECIFICITY OF CYTOCHROME P450(CAM) AND ITS L244A MUTANT

The substrate specificity of cytochrome P450, defined as the ability o f a compound to promote NAD(P)H and O-2 utilization in the production of either organic or reduced oxygen metabolites, is largely determined by steric and hydrophobic interactions. P450 specificity may therefor e be determined by the ''fit'' of a compound within the active site. A receptor-constrained three-dimensional screening program (DOCK) has b een used to select 11 compounds predicted to fit within the P450(cam) active site and 5 compounds predicted to fit within the L244A P450(cam ) but not wild-type active site. The 16 compounds were evaluated as P4 50(cam) substrates by measuring (a) binding to the enzyme, (b) stimula tion of NADH and O-2 consumption, (c) enhancement of H2O2 production, and (d) formation of organic metabolites. Seven of the compounds predi cted to fit in the active site, and none of the compounds predicted no t to fit, were found to be substrates. Compounds predicted to fit very tightly within the active site were poor or non-substrates. The L244A P450(cam) mutant was constructed, expressed, purified, and shown to r eadily oxidize some of the larger compounds incorrectly predicted to b e substrates for the wild-type enzyme. The 5 ligands selected to fit t he L244A but not wild-type sites were not detectable substrates, presu mably because they fit too tightly into the active site. Retroactive a djustments of the docking program based on an analysis of the docking parameters, particularly variation of the minimum distance allowed bet ween ligand and protein atoms, allow correct predictions for the activ ity of 15 of the 16 compounds with wild-type P450(cam). The DOCK predi ctions for the L244A mutant were also improved by changing the minimum contact distances to disfavor the larger compounds. The results indic ate that ligands that fill the; active site are marginal or non-substr ates. A degree of freedom of motion is required for substrate position ing and catalytic function. if parameters are chosen to allow for this requirement, P450(cam) substrate predictions based on Ligand docking in the active site can be reasonably accurate.