A Modified Arrhenius Approach to Thermodynamically Study Regioselectivity in Cytochrome P450‐Catalyzed Substrate Conversion

Abstract

Scheme1.Kinetic scheme for the catalytic conversion of substrate St otwo possible products, P 1 and P 2 .The associatedrate constants k cat,1 and k cat,2 dependonrate constants for the individual steps of the catalytic conversion, which comprise bindingt o( k b )a nd unbinding of (k u )t he enzyme-substrate complex ES, interconversion between ES 1 and ES 2 (k i , k -i ), formation of the enzyme-product complex EP out of ES (k p ), and unbindingoft he EP complex (k r ).

Different Geometric Requirements for Cytochrome P450-Catalyzed Aliphatic Versus Aromatic Hydroxylation Results in Chemoselective Oxidation

“…For the oxidation of 4-cyclohexylbenzoate, the ideal TS geometry features a nearly linear C–H···O array (Figure S14). Its important geometrical features are the O–H distance, C–H–O angle, and Fe–O–H angle. − Figure shows how these three features varied, for each cyclohexyl hydrogen, during 2000 ns of MD simulations. The QM ideal TS geometry is shown as a black dot in each part of the figure.…”

“…The 4-phenylbenzoate system is considered next. Previous MD analyses of P450 catalyzed aromatic oxidation employed C–O distances and Fe–O–C angles to identify potential sites of oxidation, including a recent paper published contemporaneously by Wu and coworkers investigating the hydroxylation of toluene catalyzed by CYP102A1 (P450BM3). ,− , A plot showing these two parameters for the phenyl ring carbons of 4-phenylbenzoate during our MD simulations is shown in Figure . Note that, unlike the cyclohexyl substrate discussed above, the symmetry-related carbons of the phenyl ring are given identical labels here, as their oxidation leads to identical products.…”

Computation-Aided Engineering of Cytochrome P450 for the Production of Pravastatin

“…MD has previously been applied to P450s, e.g. , to understand the conformational dynamics and substrate binding mechanism, ,− , to evaluate the enhanced activity of experimentally characterized variants, to search for positions to mutate for altering substrate specificity, , and to explain the specificity of mutants. − , Recently, MD runs of 22 ns were performed to examine the regioselectivity of steroid- and warfarin-hydroxylation, with scoring for near-attack conformations. , However, due to the high computational cost, long MD simulations that are commonly used for studying protein dynamics cannot be applied for scoring large numbers of enzyme variants, and the user is bound to a restricted search space. A ranking method with high-throughput capacity is important since Rosetta and other computational design algorithms produce large numbers of primary designs with different sequences and conformations.…”

“…Due to the marked flexibility in their active-site region and the occurrence of ligand-induced conformational changes, cytochrome P450s are challenging enzymes for the rationalization of catalytic properties by computational approaches. − Recently, short MD simulations were performed on P450s to examine selectivity in the hydroxylation of steroids, warfarin, testosterone, terpene and limonene, and other substrates. − In these examples, P450 variants were studied using nanosecond timescale MD simulations with scoring for catalytically productive conformations along MD trajectories, which explained the observed stereoselectivity. Moreover, nanosecond timescale MD on P450s was employed to report on binding properties of aflatoxin B1, caffeine, and others. − …”

“… 43 − 45 Recently, short MD simulations were performed on P450s to examine selectivity in the hydroxylation of steroids, 46 warfarin, 47 testosterone, 48 terpene and limonene, 49 and other substrates. 50 − 56 In these examples, P450 variants were studied using nanosecond timescale MD simulations with scoring for catalytically productive conformations along MD trajectories, which explained the observed stereoselectivity. Moreover, nanosecond timescale MD on P450s was employed to report on binding properties of aflatoxin B1, 57 caffeine, 58 and others.…”