Integrative Mechanistic Analysis of Essential Cytochrome P450 Reactions in Endobiotic Metabolism

Abstract

Cytochrome P450 enzymes (P450s) constitute a diverse superfamily of heme-containing monooxygenases that catalyze critical reactions in endobiotic metabolism, such as with steroids and fatty acids. Several essential steroidogenic P450 oxidations are multistep reactions that involve atypical carbon-carbon bond scission steps. The concertedness (processivity) of the multistep reactions of P450s 11A1 and 51A1 and the mechanism of C-C bond scission by five P450 51 enzymes was evaluated. The fatty acid substrates of some P450 reactions are primarily bound to fatty acid binding protein (FABP1), and whether substrate-bound FABP1 transfer substrates to lipid-metabolizing P450 4A11 was assessed. The role of FABP1 in the P450 4A11 reaction was analyzed (Chapter 3). Experimental determination of kinetic parameters (Kd, kon, koff, kcat) with both proteins revealed that the substrate (palmitate) bound 100-fold tighter to FABP1 than to P450, and FABP1 did not significantly inhibit the P450 reaction. Computational kinetic modeling of palmitate oxidation using experimental rate constants supported a direct substrate transfer mechanism from FABP1 to P450. The multistep reactions of P450s 51A1 and 11A1 were examined (Chapter 4). Synthesis of sterol intermediates and measurement of binding and oxidation kinetics of both enzymes with their corresponding sterols revealed oxidation rate > dissociation rate. Analysis of single turnover reactions indicated that reaction intermediates were short-lived (low t1/2). Both reactions were kinetically poised for final product formation – not intermediate dissociation – indicating highly processive reactions. The P450 51 C-C bond cleavage mechanism was discerned using an isotope labeling approach (Chapter 5). 18O incorporation studies into the C-C bond cleavage byproduct formic acid revealed a predominantly (~85%) nucleophilic (Compound 0) mechanism, with a minor (~15%) contribution of an electrophilic (Compound I) pathway (confirmed by complementary H218O experiments). Subsequent efforts to hone Compound 0 chemistry in P450BM-3 and P450 11A1 reactions by engineering electrophilic (“pro-Compound 0”) substrate derivatives revealed only Compound I products formed by both enzymes at slow rates. These studies together characterize mechanisms of P450 activities in endobiotic metabolism. The results are informative to future efforts in drug development, industrial biosynthetic chemistry, and biocatalyst development.