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Robert P. Hanzlik - One of the best experts on this subject based on the ideXlab platform.
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^1H high-resolution magic-angle spinning (HR-MAS) NMR analysis of ligand density on resins using a resin internal standard
Analytical and Bioanalytical Chemistry, 2004Co-Authors: Laura H. Lucas, Robert P. Hanzlik, Matthew A. Cerny, Yakov M. Koen, Cynthia K. LariveAbstract:We recently attempted to generate an affinity chromatography adsorbent to purify Cytochrome P450 4A1 by coupling 11-(1′-imidazolyl)-3,6,9-trioxaundecanoic acid to Toyopearl AF-Amino 650 M resin. Variations in ligand density for several resin batches were quantified by high-resolution magic-angle spinning (HR-MAS) NMR spectroscopy using a novel resin internal standard. The uniquely designed ImQ internal resin standard yields its signature resonance in a transparent region of the analyte spectrum making suppression of the polymer background unnecessary. This method enabled us to target a reasonable ligand density for enzyme purification and provides an advantageous alternative to quantitation against soluble standards or protonated solvent.
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Active site structure and substrate specificity of Cytochrome P450 4A1: steric control of ligand approach perpendicular to heme plane.
Biochemical and Biophysical Research Communications, 1996Co-Authors: Ramesh B. Bambal, Robert P. HanzlikAbstract:Abstract The active site of an engineered, expressed fusion protein containing the sequences of Cytochrome P450 4A1 (lauric acid ω-hydroxylase) and NADPH-Cytochrome P450 reductase, has been probed with olefinic, acetylenic, aromatic, and other analogs of its normal substrate. The fusion protein ω-hydroxylates lauric acid, epoxidizes 11-dodocenoic acid, oxidizes 11-dodecynoic acid to 1,12-dodecandioic acid, but does not oxidize 9-phenylnonanoic acid. Nevertheless, the latter is a tight-binding Type I ligand (K s = 0.77 μM) and a potent inhibitor of lauric acid hydroxylation. These and other observations are used to construct an active site model that accounts for its remarkable substrate and inhibitor specificity.
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Effects of steric bulk and conformational rigidity on fatty acid omega hydroxylation by a Cytochrome P450 4A1 fusion protein
Archives of Biochemistry and Biophysics, 1996Co-Authors: Ramesh B. Bambal, Robert P. HanzlikAbstract:Abstract The action of Cytochrome P450 4A1 (CYP4A1) on fatty acid substrates is characterized by a pronounced regioselectivity for ω-hydroxylation. To elucidate the chemical basis of this specificity we probed the active site of a CYP4A1 fusion protein (f4A1) with bulky and/or rigid analogs of lauric acid, the optimum natural substrate for f4A1 and CYP4A1. f4A1 efficiently ω-hydroxylates lauric acid, epoxidizes 11-dodecenoic acid, and oxidizes 11-dodecynoic acid to 1,12-dodecanedioic acid. Medium length fatty acids having ω-terminal groups as large as t -butyl or m -tolyloxy bind tightly to f4A1 as Type I ligands and are efficiently hydroxylated on their methyl termini. ω-Phenylnonanoic acid also induces a Type I binding spectrum ( K s = 0.77 μ m ) but fails to undergo hydroxylation and strongly inhibits lauric acid hydroxylation by f4A1. Slightly shorter acids such as ω-phenyloctanoic acid, naproxen, and ibuprofen also strongly inhibit lauric acid hydroxylation but do not induce a Type I spectrum and do not undergo hydroxylation. Like 10-methoxydecanoic acid, the rigid and rod-like 4′-methoxy-4-biphenylcarboxylic acid is O-demethylated by f4A1, which also ω-hydroxylates m - and p -heptyloxybenzoic acids but not m - or p -amyloxyhydrocinnamic acids. The histamine antagonist cimetidine and the peroxisome proliferator perfluorodecanoic acid are both potent inhibitors of f4A1. Thus the active site of f4A1 is quite tolerant of steric bulk and rigidity around the heme region and the polar group recognition site, but perhaps less so in the midchain region. Although CYP4A enzymes are not usually regarded as “drug metabolizing P450s,” the fact that commonly used therapeutic agents strongly inhibit lauric acid ω-hydroxylation by f4A1 as well as liver microsomes from clofibrate-induded rats suggests these and related agents could potentially interfere with the contribution of CYP4A enzymes to the metabolism of endogenous lipids.
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fatty acid discrimination and omega hydroxylation by Cytochrome P450 4A1 and a Cytochrome P4504A1 nadph P450 reductase fusion protein
Archives of Biochemistry and Biophysics, 1995Co-Authors: Michail A. Alterman, Chandra S. Chaurasia, James P. Hardwick, Robert P. HanzlikAbstract:The ω-hydroxylation of fatty acids by certain Cytochrome P450 enzymes shows a degree of chain-length and regiospecificity which is remarkable in view of the conformational flexibility of these substrates, the strong similarity in properties among homologs, and the lack of polar groups (other than the carboxy terminus) with which to guide and strengthen enzyme-substrate interactions. To investigate the chemical basis for these features of ω-hydroxylation we designed and synthesized a series of lauric acid analogs and evaluated them as substrates and inhibitors of ω-hydroxylation catalyzed by Cytochrome P4504A1 and a Cytochrome P450 4A1/NADPH-P450 reductase fusion protein. Among n-alkanoic acids, lauric acid was found to have the optimum chain length for the fusion protein, as it does for native Cytochrome P450 4A1. With both enzymes, chain shortening caused a precipitous drop in turnover while chain lengthening caused a gradual drop in turnover. The fusion protein ω-hydroxylated methyl laurate and lauryl alcohol about 110th as efficiently as lauric acid, but it did not hydroxylate lauramide. 10-Methoxydecanoic acid underwent O-demethylation (via ω-hydroxylation). The branched substrate 11-methyllauric acid was hydroxylated efficiently and selectively at the ω-position. In contrast, the cyclopropyl analog 11,12-methanolauric acid was not detectably hydroxylated, although it induced Type I binding spectrum and inhibited lauric acid ω-hydroxylation by 43% at equimolar concentrations. ω-(Imidazolyl)-decanoic acid induced a Type II heme-binding spectrum and was an especially potent inhibitor of lauric acid hydroxylation. Collectively these data suggest that the active site of Cytochrome P450 4A1 has an elongated tubular shape of definite length (ca. 14 A) with a recognition site for polar groups (including but not limited to carboxyl) at its entrance and the (oxo)heme group at its terminus.
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FATTY ACID DISCRIMINATION AND OMEGA -HYDROXYLATION BY Cytochrome P450 4A1 AND A Cytochrome P4504A1/NADPH-P450 REDUCTASE FUSION PROTEIN
Archives of Biochemistry and Biophysics, 1995Co-Authors: Michail A. Alterman, Chandra S. Chaurasia, James P. Hardwick, Robert P. HanzlikAbstract:The ω-hydroxylation of fatty acids by certain Cytochrome P450 enzymes shows a degree of chain-length and regiospecificity which is remarkable in view of the conformational flexibility of these substrates, the strong similarity in properties among homologs, and the lack of polar groups (other than the carboxy terminus) with which to guide and strengthen enzyme-substrate interactions. To investigate the chemical basis for these features of ω-hydroxylation we designed and synthesized a series of lauric acid analogs and evaluated them as substrates and inhibitors of ω-hydroxylation catalyzed by Cytochrome P4504A1 and a Cytochrome P450 4A1/NADPH-P450 reductase fusion protein. Among n-alkanoic acids, lauric acid was found to have the optimum chain length for the fusion protein, as it does for native Cytochrome P450 4A1. With both enzymes, chain shortening caused a precipitous drop in turnover while chain lengthening caused a gradual drop in turnover. The fusion protein ω-hydroxylated methyl laurate and lauryl alcohol about 110th as efficiently as lauric acid, but it did not hydroxylate lauramide. 10-Methoxydecanoic acid underwent O-demethylation (via ω-hydroxylation). The branched substrate 11-methyllauric acid was hydroxylated efficiently and selectively at the ω-position. In contrast, the cyclopropyl analog 11,12-methanolauric acid was not detectably hydroxylated, although it induced Type I binding spectrum and inhibited lauric acid ω-hydroxylation by 43% at equimolar concentrations. ω-(Imidazolyl)-decanoic acid induced a Type II heme-binding spectrum and was an especially potent inhibitor of lauric acid hydroxylation. Collectively these data suggest that the active site of Cytochrome P450 4A1 has an elongated tubular shape of definite length (ca. 14 A) with a recognition site for polar groups (including but not limited to carboxyl) at its entrance and the (oxo)heme group at its terminus.
Michail A. Alterman - One of the best experts on this subject based on the ideXlab platform.
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fatty acid discrimination and omega hydroxylation by Cytochrome P450 4A1 and a Cytochrome P4504A1 nadph P450 reductase fusion protein
Archives of Biochemistry and Biophysics, 1995Co-Authors: Michail A. Alterman, Chandra S. Chaurasia, James P. Hardwick, Robert P. HanzlikAbstract:The ω-hydroxylation of fatty acids by certain Cytochrome P450 enzymes shows a degree of chain-length and regiospecificity which is remarkable in view of the conformational flexibility of these substrates, the strong similarity in properties among homologs, and the lack of polar groups (other than the carboxy terminus) with which to guide and strengthen enzyme-substrate interactions. To investigate the chemical basis for these features of ω-hydroxylation we designed and synthesized a series of lauric acid analogs and evaluated them as substrates and inhibitors of ω-hydroxylation catalyzed by Cytochrome P4504A1 and a Cytochrome P450 4A1/NADPH-P450 reductase fusion protein. Among n-alkanoic acids, lauric acid was found to have the optimum chain length for the fusion protein, as it does for native Cytochrome P450 4A1. With both enzymes, chain shortening caused a precipitous drop in turnover while chain lengthening caused a gradual drop in turnover. The fusion protein ω-hydroxylated methyl laurate and lauryl alcohol about 110th as efficiently as lauric acid, but it did not hydroxylate lauramide. 10-Methoxydecanoic acid underwent O-demethylation (via ω-hydroxylation). The branched substrate 11-methyllauric acid was hydroxylated efficiently and selectively at the ω-position. In contrast, the cyclopropyl analog 11,12-methanolauric acid was not detectably hydroxylated, although it induced Type I binding spectrum and inhibited lauric acid ω-hydroxylation by 43% at equimolar concentrations. ω-(Imidazolyl)-decanoic acid induced a Type II heme-binding spectrum and was an especially potent inhibitor of lauric acid hydroxylation. Collectively these data suggest that the active site of Cytochrome P450 4A1 has an elongated tubular shape of definite length (ca. 14 A) with a recognition site for polar groups (including but not limited to carboxyl) at its entrance and the (oxo)heme group at its terminus.
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FATTY ACID DISCRIMINATION AND OMEGA -HYDROXYLATION BY Cytochrome P450 4A1 AND A Cytochrome P4504A1/NADPH-P450 REDUCTASE FUSION PROTEIN
Archives of Biochemistry and Biophysics, 1995Co-Authors: Michail A. Alterman, Chandra S. Chaurasia, James P. Hardwick, Robert P. HanzlikAbstract:The ω-hydroxylation of fatty acids by certain Cytochrome P450 enzymes shows a degree of chain-length and regiospecificity which is remarkable in view of the conformational flexibility of these substrates, the strong similarity in properties among homologs, and the lack of polar groups (other than the carboxy terminus) with which to guide and strengthen enzyme-substrate interactions. To investigate the chemical basis for these features of ω-hydroxylation we designed and synthesized a series of lauric acid analogs and evaluated them as substrates and inhibitors of ω-hydroxylation catalyzed by Cytochrome P4504A1 and a Cytochrome P450 4A1/NADPH-P450 reductase fusion protein. Among n-alkanoic acids, lauric acid was found to have the optimum chain length for the fusion protein, as it does for native Cytochrome P450 4A1. With both enzymes, chain shortening caused a precipitous drop in turnover while chain lengthening caused a gradual drop in turnover. The fusion protein ω-hydroxylated methyl laurate and lauryl alcohol about 110th as efficiently as lauric acid, but it did not hydroxylate lauramide. 10-Methoxydecanoic acid underwent O-demethylation (via ω-hydroxylation). The branched substrate 11-methyllauric acid was hydroxylated efficiently and selectively at the ω-position. In contrast, the cyclopropyl analog 11,12-methanolauric acid was not detectably hydroxylated, although it induced Type I binding spectrum and inhibited lauric acid ω-hydroxylation by 43% at equimolar concentrations. ω-(Imidazolyl)-decanoic acid induced a Type II heme-binding spectrum and was an especially potent inhibitor of lauric acid hydroxylation. Collectively these data suggest that the active site of Cytochrome P450 4A1 has an elongated tubular shape of definite length (ca. 14 A) with a recognition site for polar groups (including but not limited to carboxyl) at its entrance and the (oxo)heme group at its terminus.
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Heteroatom Substitution Shifts Regioselectivity of Lauric Acid Metabolism from ω-Hydroxylation to (ω-1)-Oxidation
Biochemical and Biophysical Research Communications, 1995Co-Authors: Michail A. Alterman, Chandra S. Chaurasia, Robert P. HanzlikAbstract:Abstract In contrast to several isofoms of Cytochrome P450 (including 1A1, 1A2, 2B1, 2C2, 2C6 and 2E1), Cytochrome P450 4A1 and a fusion protein derived from it, show a strong preference for hydroxylation of lauric acid (C 12 ) at the less chemically reactive ω-CH 3 group instead of the more reactive (ω-1)-CH 2 group. We have explored the interplay of steric effects on substrate binding vs chemical reactivity at various substrate loci in determining the striking difference in regioselectivity of CYP2B1 and a 4A1-derived fusion protein, through studies with heteroatom substituted analogs of lauric acid, i.e.,10-methoxydecanoic acid and 10-methylthiodecanoic acid. With both enzymes the former undergoes simple ω-hydroxylation (giving 10-hydroxydecanoic acid and HCHO), but the latter undergoes facile S-oxidation at the ω-1 position instead of ω-hydroxylation. The dramatic shift in the regioselectivity of the fusion protein toward the thia analog is consistent with the greater length of C-S bonds and the greater atomic radius and polarizability of sulfur lone-pair electrons within an otherwise restrictive active site.
Slawomir Pikula - One of the best experts on this subject based on the ideXlab platform.
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10-Undecynoic acid, an inhibitor of Cytochrome P450 4A1, inhibits ethanolamine-specific phospholipid base exchange reaction in rat liver microsomes.
Acta Biochimica Polonica, 1999Co-Authors: Jacek Lenart, Slawomir PikulaAbstract:1,12-Dodecanedioic acid, the end-product of omega-hydroxylation of lauric acid, stimulates in a concentration dependent manner, phosphatidylethanolamine synthesis via ethanolamine-specific phospholipid base exchange reaction in rat liver endoplasmic reticulum. On the other hand, administration to rats of 10-undecynoic acid, a specific inhibitor of omega-hydroxylation reaction catalyzed by Cytochrome P450 4A1, inhibits the ethanolamine-specific phospholipid base exchange activity by 30%. This is accompanied by a small but significant decrease in phosphatidylethanolamine content in the endoplasmic reticulum and inhibition of Cytochrome P450 4A1. On the basis of these results it can be proposed that a functional relationship between Cytochrome P450 4A1 and phosphatidylethanolamine synthesis exists in rat liver. Cytochrome P450 4A1 modulates the cellular level of lauric acid, an inhibitor of phospholipid synthesis. In turn, ethanolamine-specific phospholipid base exchange reaction provides molecular species of phospholipids, containing mainly long-chain polyunsaturated fatty acid moieties, required for the optimal activity of Cytochrome P450 4A1.
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The induction of Cytochrome P450 isoform, CYP4A1, by clofibrate coincides with activation of ethanolamine-specific phospholipid base exchange reaction in rat liver microsomes.
Acta Biochimica Polonica, 1998Co-Authors: Jacek Lenart, Izabela Komańska, Renata Jasińska, Slawomir PikulaAbstract:Administration of a hypolipidaemic drug, clofibrate, to rats resulted, 24 h after a single intraperitoneal injection (250 mg/kg body weight), in pronounced enhancement of the rate of phosphatidylethanolamine (PE) synthesis via the PE-specific base exchange (PEBE) reaction in liver microsomes. This was accompanied by 3.4-fold activation of microsomal omega-hydroxylation of lauric acid by Cytochrome P450 4A1 isoform (CYP4A1) and an increase in the protein content of this isoform in endoplasmic reticulum (ER) membranes. Since PE represents a class of phospholipids (PL) prerequisite for proper functioning of CYP4A1, and the PEBE reaction is an inducible pathway of PL synthesis in hepatocytes under metabolic stress, one may speculate that this reaction is switched on when extensive remodelling of PL molecular species or/and massive synthesis of lipid bilayer components for membrane assembly is required.
Chandra S. Chaurasia - One of the best experts on this subject based on the ideXlab platform.
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fatty acid discrimination and omega hydroxylation by Cytochrome P450 4A1 and a Cytochrome P4504A1 nadph P450 reductase fusion protein
Archives of Biochemistry and Biophysics, 1995Co-Authors: Michail A. Alterman, Chandra S. Chaurasia, James P. Hardwick, Robert P. HanzlikAbstract:The ω-hydroxylation of fatty acids by certain Cytochrome P450 enzymes shows a degree of chain-length and regiospecificity which is remarkable in view of the conformational flexibility of these substrates, the strong similarity in properties among homologs, and the lack of polar groups (other than the carboxy terminus) with which to guide and strengthen enzyme-substrate interactions. To investigate the chemical basis for these features of ω-hydroxylation we designed and synthesized a series of lauric acid analogs and evaluated them as substrates and inhibitors of ω-hydroxylation catalyzed by Cytochrome P4504A1 and a Cytochrome P450 4A1/NADPH-P450 reductase fusion protein. Among n-alkanoic acids, lauric acid was found to have the optimum chain length for the fusion protein, as it does for native Cytochrome P450 4A1. With both enzymes, chain shortening caused a precipitous drop in turnover while chain lengthening caused a gradual drop in turnover. The fusion protein ω-hydroxylated methyl laurate and lauryl alcohol about 110th as efficiently as lauric acid, but it did not hydroxylate lauramide. 10-Methoxydecanoic acid underwent O-demethylation (via ω-hydroxylation). The branched substrate 11-methyllauric acid was hydroxylated efficiently and selectively at the ω-position. In contrast, the cyclopropyl analog 11,12-methanolauric acid was not detectably hydroxylated, although it induced Type I binding spectrum and inhibited lauric acid ω-hydroxylation by 43% at equimolar concentrations. ω-(Imidazolyl)-decanoic acid induced a Type II heme-binding spectrum and was an especially potent inhibitor of lauric acid hydroxylation. Collectively these data suggest that the active site of Cytochrome P450 4A1 has an elongated tubular shape of definite length (ca. 14 A) with a recognition site for polar groups (including but not limited to carboxyl) at its entrance and the (oxo)heme group at its terminus.
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FATTY ACID DISCRIMINATION AND OMEGA -HYDROXYLATION BY Cytochrome P450 4A1 AND A Cytochrome P4504A1/NADPH-P450 REDUCTASE FUSION PROTEIN
Archives of Biochemistry and Biophysics, 1995Co-Authors: Michail A. Alterman, Chandra S. Chaurasia, James P. Hardwick, Robert P. HanzlikAbstract:The ω-hydroxylation of fatty acids by certain Cytochrome P450 enzymes shows a degree of chain-length and regiospecificity which is remarkable in view of the conformational flexibility of these substrates, the strong similarity in properties among homologs, and the lack of polar groups (other than the carboxy terminus) with which to guide and strengthen enzyme-substrate interactions. To investigate the chemical basis for these features of ω-hydroxylation we designed and synthesized a series of lauric acid analogs and evaluated them as substrates and inhibitors of ω-hydroxylation catalyzed by Cytochrome P4504A1 and a Cytochrome P450 4A1/NADPH-P450 reductase fusion protein. Among n-alkanoic acids, lauric acid was found to have the optimum chain length for the fusion protein, as it does for native Cytochrome P450 4A1. With both enzymes, chain shortening caused a precipitous drop in turnover while chain lengthening caused a gradual drop in turnover. The fusion protein ω-hydroxylated methyl laurate and lauryl alcohol about 110th as efficiently as lauric acid, but it did not hydroxylate lauramide. 10-Methoxydecanoic acid underwent O-demethylation (via ω-hydroxylation). The branched substrate 11-methyllauric acid was hydroxylated efficiently and selectively at the ω-position. In contrast, the cyclopropyl analog 11,12-methanolauric acid was not detectably hydroxylated, although it induced Type I binding spectrum and inhibited lauric acid ω-hydroxylation by 43% at equimolar concentrations. ω-(Imidazolyl)-decanoic acid induced a Type II heme-binding spectrum and was an especially potent inhibitor of lauric acid hydroxylation. Collectively these data suggest that the active site of Cytochrome P450 4A1 has an elongated tubular shape of definite length (ca. 14 A) with a recognition site for polar groups (including but not limited to carboxyl) at its entrance and the (oxo)heme group at its terminus.
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Heteroatom Substitution Shifts Regioselectivity of Lauric Acid Metabolism from ω-Hydroxylation to (ω-1)-Oxidation
Biochemical and Biophysical Research Communications, 1995Co-Authors: Michail A. Alterman, Chandra S. Chaurasia, Robert P. HanzlikAbstract:Abstract In contrast to several isofoms of Cytochrome P450 (including 1A1, 1A2, 2B1, 2C2, 2C6 and 2E1), Cytochrome P450 4A1 and a fusion protein derived from it, show a strong preference for hydroxylation of lauric acid (C 12 ) at the less chemically reactive ω-CH 3 group instead of the more reactive (ω-1)-CH 2 group. We have explored the interplay of steric effects on substrate binding vs chemical reactivity at various substrate loci in determining the striking difference in regioselectivity of CYP2B1 and a 4A1-derived fusion protein, through studies with heteroatom substituted analogs of lauric acid, i.e.,10-methoxydecanoic acid and 10-methylthiodecanoic acid. With both enzymes the former undergoes simple ω-hydroxylation (giving 10-hydroxydecanoic acid and HCHO), but the latter undergoes facile S-oxidation at the ω-1 position instead of ω-hydroxylation. The dramatic shift in the regioselectivity of the fusion protein toward the thia analog is consistent with the greater length of C-S bonds and the greater atomic radius and polarizability of sulfur lone-pair electrons within an otherwise restrictive active site.
James P. Hardwick - One of the best experts on this subject based on the ideXlab platform.
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fatty acid discrimination and omega hydroxylation by Cytochrome P450 4A1 and a Cytochrome P4504A1 nadph P450 reductase fusion protein
Archives of Biochemistry and Biophysics, 1995Co-Authors: Michail A. Alterman, Chandra S. Chaurasia, James P. Hardwick, Robert P. HanzlikAbstract:The ω-hydroxylation of fatty acids by certain Cytochrome P450 enzymes shows a degree of chain-length and regiospecificity which is remarkable in view of the conformational flexibility of these substrates, the strong similarity in properties among homologs, and the lack of polar groups (other than the carboxy terminus) with which to guide and strengthen enzyme-substrate interactions. To investigate the chemical basis for these features of ω-hydroxylation we designed and synthesized a series of lauric acid analogs and evaluated them as substrates and inhibitors of ω-hydroxylation catalyzed by Cytochrome P4504A1 and a Cytochrome P450 4A1/NADPH-P450 reductase fusion protein. Among n-alkanoic acids, lauric acid was found to have the optimum chain length for the fusion protein, as it does for native Cytochrome P450 4A1. With both enzymes, chain shortening caused a precipitous drop in turnover while chain lengthening caused a gradual drop in turnover. The fusion protein ω-hydroxylated methyl laurate and lauryl alcohol about 110th as efficiently as lauric acid, but it did not hydroxylate lauramide. 10-Methoxydecanoic acid underwent O-demethylation (via ω-hydroxylation). The branched substrate 11-methyllauric acid was hydroxylated efficiently and selectively at the ω-position. In contrast, the cyclopropyl analog 11,12-methanolauric acid was not detectably hydroxylated, although it induced Type I binding spectrum and inhibited lauric acid ω-hydroxylation by 43% at equimolar concentrations. ω-(Imidazolyl)-decanoic acid induced a Type II heme-binding spectrum and was an especially potent inhibitor of lauric acid hydroxylation. Collectively these data suggest that the active site of Cytochrome P450 4A1 has an elongated tubular shape of definite length (ca. 14 A) with a recognition site for polar groups (including but not limited to carboxyl) at its entrance and the (oxo)heme group at its terminus.
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FATTY ACID DISCRIMINATION AND OMEGA -HYDROXYLATION BY Cytochrome P450 4A1 AND A Cytochrome P4504A1/NADPH-P450 REDUCTASE FUSION PROTEIN
Archives of Biochemistry and Biophysics, 1995Co-Authors: Michail A. Alterman, Chandra S. Chaurasia, James P. Hardwick, Robert P. HanzlikAbstract:The ω-hydroxylation of fatty acids by certain Cytochrome P450 enzymes shows a degree of chain-length and regiospecificity which is remarkable in view of the conformational flexibility of these substrates, the strong similarity in properties among homologs, and the lack of polar groups (other than the carboxy terminus) with which to guide and strengthen enzyme-substrate interactions. To investigate the chemical basis for these features of ω-hydroxylation we designed and synthesized a series of lauric acid analogs and evaluated them as substrates and inhibitors of ω-hydroxylation catalyzed by Cytochrome P4504A1 and a Cytochrome P450 4A1/NADPH-P450 reductase fusion protein. Among n-alkanoic acids, lauric acid was found to have the optimum chain length for the fusion protein, as it does for native Cytochrome P450 4A1. With both enzymes, chain shortening caused a precipitous drop in turnover while chain lengthening caused a gradual drop in turnover. The fusion protein ω-hydroxylated methyl laurate and lauryl alcohol about 110th as efficiently as lauric acid, but it did not hydroxylate lauramide. 10-Methoxydecanoic acid underwent O-demethylation (via ω-hydroxylation). The branched substrate 11-methyllauric acid was hydroxylated efficiently and selectively at the ω-position. In contrast, the cyclopropyl analog 11,12-methanolauric acid was not detectably hydroxylated, although it induced Type I binding spectrum and inhibited lauric acid ω-hydroxylation by 43% at equimolar concentrations. ω-(Imidazolyl)-decanoic acid induced a Type II heme-binding spectrum and was an especially potent inhibitor of lauric acid hydroxylation. Collectively these data suggest that the active site of Cytochrome P450 4A1 has an elongated tubular shape of definite length (ca. 14 A) with a recognition site for polar groups (including but not limited to carboxyl) at its entrance and the (oxo)heme group at its terminus.
