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James R. Halpert - One of the best experts on this subject based on the ideXlab platform.
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Use of chimeric enzymes and site-directed mutagenesis for identification of three key residues responsible for differences in Steroid Hydroxylation between canine cytochromes P-450 3A12 and 3A26.
Molecular pharmacology, 1999Co-Authors: David J. Fraser, Greg R. Harlow, James R. HalpertAbstract:Canine cytochromes P-450 3A12 and 3A26 differ by 22 out of 503 amino acid residues. Chimeric constructs and site-directed mutants were used to identify the residues responsible for the much higher rates of Steroid Hydroxylation by 3A12. Six initial 3A12/3A26 hybrids were generated using convenient restriction sites, and site-directed mutagenesis was used to restore full 3A12 activity to two of the hybrids. One pair of 3A12/3A26 chimeras indicated that the first four residue differences between 3A12 and 3A26 were at least partially responsible for the differences in progesterone Hydroxylation. Conversion in one of the hybrids of the Ile-187 residue found in 3A26 to the Thr in 3A12 conferred 3A12 levels of progesterone 6beta-hydroxylase activity. Analysis of another chimera identified key residues within an internal PstI fragment (codons 331-459) containing six amino acid residue differences. Subsequent site-directed mutagenesis of 3A26 residues Ser-368 and Val-369 to Pro and Ile, respectively, restored the rate of formation of 6beta-hydroxyprogesterone by the hybrid to that of 3A12. The simultaneous conversion of 3A26 residues 187, 368, and 369 to those of 3A12 conferred greater than a third of the progesterone 6beta-hydroxylase activity and all of the testosterone and androstenedione 6beta-hydroxylase activity of 3A12. Addition of the carboxyl terminal 44 3A12 residues to the 3A26 triple mutant doubled progesterone 6beta-hydroxylase activity. This is the first study to use catalytically distinct cytochromes P-450 3A from the same species in the elucidation of structure-function relationships.
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use of chimeric enzymes and site directed mutagenesis for identification of three key residues responsible for differences in Steroid Hydroxylation between canine cytochromes p 450 3a12 and 3a26
Molecular Pharmacology, 1999Co-Authors: David J. Fraser, Greg R. Harlow, James R. HalpertAbstract:Canine cytochromes P-450 3A12 and 3A26 differ by 22 out of 503 amino acid residues. Chimeric constructs and site-directed mutants were used to identify the residues responsible for the much higher rates of Steroid Hydroxylation by 3A12. Six initial 3A12/3A26 hybrids were generated using convenient restriction sites, and site-directed mutagenesis was used to restore full 3A12 activity to two of the hybrids. One pair of 3A12/3A26 chimeras indicated that the first four residue differences between 3A12 and 3A26 were at least partially responsible for the differences in progesterone Hydroxylation. Conversion in one of the hybrids of the Ile-187 residue found in 3A26 to the Thr in 3A12 conferred 3A12 levels of progesterone 6β-hydroxylase activity. Analysis of another chimera identified key residues within an internal Pst I fragment (codons 331–459) containing six amino acid residue differences. Subsequent site-directed mutagenesis of 3A26 residues Ser-368 and Val-369 to Pro and Ile, respectively, restored the rate of formation of 6β-hydroxyprogesterone by the hybrid to that of 3A12. The simultaneous conversion of 3A26 residues 187, 368, and 369 to those of 3A12 conferred greater than a third of the progesterone 6β-hydroxylase activity and all of the testosterone and androstenedione 6β-hydroxylase activity of 3A12. Addition of the carboxyl terminal 44 3A12 residues to the 3A26 triple mutant doubled progesterone 6β-hydroxylase activity. This is the first study to use catalytically distinct cytochromes P-450 3A from the same species in the elucidation of structure-function relationships.
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analysis of human cytochrome p450 3a4 cooperativity construction and characterization of a site directed mutant that displays hyperbolic Steroid Hydroxylation kinetics
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Greg R. Harlow, James R. HalpertAbstract:Cytochrome P450 3A4 is generally considered to be the most important human drug-metabolizing enzyme and is known to catalyze the oxidation of a number of substrates in a cooperative manner. An allosteric mechanism is usually invoked to explain the cooperativity. Based on a structure–activity study from another laboratory using various effector–substrate combinations and on our own studies using site-directed mutagenesis and computer modeling of P450 3A4, the most likely location of effector binding is in the active site along with the substrate. Our study was designed to test this hypothesis by replacing residues Leu-211 and Asp-214 with the larger Phe and Glu, respectively. These residues were predicted to constitute a portion of the effector binding site, and the substitutions were designed to mimic the action of the effector by reducing the size of the active site. The L211F/D214E double mutant displayed an increased rate of testosterone and progesterone 6β-Hydroxylation at low substrate concentrations and a decreased level of heterotropic stimulation elicited by α-naphthoflavone. Kinetic analyses of the double mutant revealed the absence of homotropic cooperativity with either Steroid substrate. At low substrate concentrations the Steroid 6β-hydroxylase activity of the wild-type enzyme was stimulated by a second Steroid, whereas L211F/D214E displayed simple substrate inhibition. To analyze L211F/D214E at a more mechanistic level, spectral binding studies were carried out. Testosterone binding by the wild-type enzyme displayed homotropic cooperativity, whereas substrate binding by L211F/D214E displayed hyperbolic behavior.
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Identification of three key residues in substrate recognition site 5 of human cytochrome P450 3A4 by cassette and site-directed mutagenesis.
Biochemistry, 1997Co-Authors: Grazyna D. Szklarz, James R. HalpertAbstract:Cassette mutagenesis and site-directed mutagenesis were used to investigate the importance of individual amino acid residues at positions 364-377 of cytochrome P450 3A4 in determining Steroid Hydroxylation or stimulation by alpha-naphthoflavone. The mutants were expressed in an Escherichia coli system, and solubilized membranes were prepared. All mutants except R365G and R365K exhibited anti-3A immunoreactivity on Western blotting, although R372S and R375K were not detected as the Fe2+-CO complex. Replacement of Arg-372 by Lys yielded a typical P450 spectrum. The results indicate that the highly conserved Arg residues at positions 365 and 375 may play a role in stabilizing the tertiary structure or in heme binding. Catalytic activities of 12 mutants were examined using progesterone and testosterone as substrates, and residues 369, 370, and 373 were found to play an important role in determining substrate specificity. Although the three mutants hydroxylated progesterone and testosterone primarily at the 6beta-position like the wild-type, replacement of Ile-369 by Val suppressed progesterone 16alpha-hydroxylase activity, whereas substitution of Ala-370 with Val enhanced progesterone 16alpha-Hydroxylation. Interestingly, substitution of Leu-373 with His resulted in production of a new metabolite from both Steroids. Moreover, the mutants at positions 369 and 373 were more and less responsive, respectively, than the wild-type to alpha-naphthoflavone stimulation. Alterations in activities or expression of several mutants were interpreted using a three-dimensional model of P450 3A4. The results suggest that analogy with mammalian family 2 and bacterial cytochromes P450 can be used to predict P450 3A residues that contribute to regiospecific Steroid Hydroxylation.
Rita Bernhardt - One of the best experts on this subject based on the ideXlab platform.
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Structure-based engineering of Steroidogenic CYP260A1 for stereo- and regioselective Hydroxylation of progesterone
ACS chemical biology, 2018Co-Authors: Yogan Khatri, Ilona K. Jóźwik, Michael Ringle, Irina Alexandra Ionescu, Martin Litzenburger, Michael C. Hutter, Andy-mark W. H. Thunnissen, Rita BernhardtAbstract:The production of regio- and stereoselectively hydroxylated Steroids is of high pharmaceutical interest and can be achieved by cytochrome P450-based biocatalysts. CYP260A1 from Sorangium cellulosum strain So ce56 catalyzes Hydroxylation of C19 or C21 Steroids at the very unique 1α-position. However, the conversion of progesterone (PROG) by CYP260A1 is very unselective. In order to improve its selectivity we applied a semirational protein engineering approach, resulting in two different, highly regio- and stereoselective mutants by replacing a single serine residue (S276) of the substrate recognition site 5 with an asparagine or isoleucine. The S276N mutant converted PROG predominantly into 1α-hydroxy-PROG, while the S276I mutant led to 17α-hydroxy-PROG. We solved the high-resolution crystal structures of the PROG-bound S276N and S276I mutants, which revealed two different binding modes of PROG in the active site. The orientations were consistent with the exclusive 1α- (pro-1α binding mode) and 17α-Hydroxylation (pro-17α-binding mode) of S276N and S276I, respectively. We observed that water-mediated hydrogen bonds contribute to the stabilization of the polar C3 and C17 substituents of PROG. Both binding modes of PROG may be stabilized in the wild-type enzyme. The change in regioselectivity is mainly driven by destabilizing the alternative binding mode due to steric hindrance and hydrogen bond disruption, caused by the mutations of Ser276. Thus, for the first time, the change in the selectivity of cytochrome P450-mediated Steroid Hydroxylation created by rational mutagenesis can be explained by the obtained 3D structures of the substrate-bound mutants, providing the basis for further experiments to engineer the biocatalyst toward novel Steroid Hydroxylation positions.
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Structure-Based Engineering of Steroidogenic CYP260A1 for Stereo- and Regioselective Hydroxylation of Progesterone
2018Co-Authors: Yogan Khatri, Michael Ringle, Irina Alexandra Ionescu, Martin Litzenburger, Michael C. Hutter, Andy-mark W. H. Thunnissen, Ilona K. Jóźwik, Rita BernhardtAbstract:The production of regio- and stereoselectively hydroxylated Steroids is of high pharmaceutical interest and can be achieved by cytochrome P450-based biocatalysts. CYP260A1 from Sorangium cellulosum strain So ce56 catalyzes Hydroxylation of C19 or C21 Steroids at the very unique 1α-position. However, the conversion of progesterone (PROG) by CYP260A1 is very unselective. In order to improve its selectivity we applied a semirational protein engineering approach, resulting in two different, highly regio- and stereoselective mutants by replacing a single serine residue (S276) of the substrate recognition site 5 with an asparagine or isoleucine. The S276N mutant converted PROG predominantly into 1α-hydroxy-PROG, while the S276I mutant led to 17α-hydroxy-PROG. We solved the high-resolution crystal structures of the PROG-bound S276N and S276I mutants, which revealed two different binding modes of PROG in the active site. The orientations were consistent with the exclusive 1α- (pro-1α binding mode) and 17α-Hydroxylation (pro-17α-binding mode) of S276N and S276I, respectively. We observed that water-mediated hydrogen bonds contribute to the stabilization of the polar C3 and C17 substituents of PROG. Both binding modes of PROG may be stabilized in the wild-type enzyme. The change in regioselectivity is mainly driven by destabilizing the alternative binding mode due to steric hindrance and hydrogen bond disruption, caused by the mutations of Ser276. Thus, for the first time, the change in the selectivity of cytochrome P450-mediated Steroid Hydroxylation created by rational mutagenesis can be explained by the obtained 3D structures of the substrate-bound mutants, providing the basis for further experiments to engineer the biocatalyst toward novel Steroid Hydroxylation positions
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Mammalian and Bacterial Cytochromes P450 Involved in Steroid Hydroxylation: Regulation of Catalysis and Selectivity, and Potential Applications
Fifty Years of Cytochrome P450 Research, 2014Co-Authors: Rita BernhardtAbstract:In this review, recent results concerning the function and potential applications of mammalian Steroid hydroxylase s, which catalyze the important tailoring of Steroid molecules, are discussed. A better understanding of the mechanism and modulation of these enzymes opens up new perspectives and innovative possibilities for the treatment of diseases caused by misfunction of the Steroidogenic enzymes such as overproduction of aldosterone leading to hypertension and congestive heart failure. In this chapter, special attention is given to the role of protein–protein interactions on the activity of mitochondrial Steroid hydroxylase systems. In addition, the role of Steroids themselves as important drugs is considered. The progress in recombinant protein expression and in genome sequencing (leading to the identification of novel cytochrome P450 systems) as well as the application of engineering of mammalian and bacterial Steroid hydroxylase s opens up a tremendous reservoir of possibilities for the application of the corresponding strains and enzymes for the sustainable production of Steroidal drugs and products. It can be expected that the extensive use of methods of both enzyme and strain engineering will further promote and increase the application of Steroid hydroxylase s in biotechnological processes.
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The human Steroid hydroxylases CYP1B1 and CYP11B2.
Biological chemistry, 2002Co-Authors: Matthias Bureik, Michael Lisurek, Rita BernhardtAbstract:Major advances have been made during the last decade in our understanding of adrenal Steroid hormone biosynthesis. Two key players in these pathways are the human mitochondrial cytochrome P450 enzymes CYP11B1 and CYP11B2, which catalyze the final steps in the biosynthesis of cortisol and aldosterone. Using data from mutations found in patients suffering from Steroid hormone-related diseases, from mutagenesis studies and from the construction of three-dimensional models of these enzymes, structural information could be deduced that provide a clue to the stereo- and regiospecific Steroid Hydroxylation reactions carried out by these enzymes. In this review, we summarize the current knowledge on the physiological function and the biochemistry of these enzymes. Furthermore, the pharmacological and toxicological importance of these Steroid hydroxylases, the means for the identification of their potential inhibitors and possible biotechnological applications are discussed.
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MODULATION OF Steroid HYDROXYLASE ACTIVITY IN STABLY TRANSFECTED V79MZH11B1 AND V79MZH11B2 CELLS BY PKC AND PKD INHIBITORS
Endocrine research, 2002Co-Authors: Matthias Bureik, Annette Zeeh, Rita BernhardtAbstract:We recently observed that treatment of CYP11B2-expressing COS-1 cells with the broad range kinase inhibitor, staurosporine (STS), strongly inhibited aldosterone biosynthesis, indicating that the activity of a kinase might be a prerequisite for Steroid hydroxylase activity. In an attempt to identify such kinases, we measured conversion of 11-deoxycortisol (RSS) and 11-deoxycorticosterone (DOC) by V79MZh11B1 and V79MZh11B2 cells, respectively, in the presence of STS and also after treatment with the kinase inhibitors chelerythrine, rottlerin and Go 6976. The conversion of both substrates by both cell lines was affected in a selective manner by the kinase inhibitors, suggesting that the activity of the novel PKC-δ and either of conventional PKCs or of PKD alter Steroid Hydroxylation activity, with their influence depending on both the cytochrome P450 tested and on its Steroid substrate.
Paul B Reese - One of the best experts on this subject based on the ideXlab platform.
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Steroid Hydroxylation by whetzelinia sclerotiorum phanerochaete chrysosporium and mucor plumbeus
Steroids, 2007Co-Authors: Andrew S Lamm, Avril R M Chen, William F Reynolds, Paul B ReeseAbstract:Abstract The fungi Whetzelinia sclerotiorum ATCC 18687, Phanerochaete chrysosporium ATCC 24725 and Mucor plumbeus ATCC 4740 were examined for their ability to perform Steroid biotransformations under single phase, pulse feed conditions. The Steroids 3β-hydroxyandrost-5-en-17-one (dehydroepiandrosterone) (1), 17β-hydroxyandrost-4-en-3-one (testosterone) (5), 3β-hydroxypregn-5-en-20-one (pregnenolone) (3), pregn-4-ene-3,20-dione (progesterone) (9), 17α,21-dihydroxypregn-4-ene-3,11,20-trione (cortisone) (11), 17α,21-dihydroxypregna-1,4-diene-3,11,20-trione (prednisone) (14), and 3-hydroxyestra-1,3,5(10)-trien-17-one (estrone) (15) were fed to each fungus. The production of a number of novel metabolites is reported. Of the fungi investigated W. sclerotiorum performed the most interesting biotransformations and had a clear propensity for 2β, 6β/7β and 15β/16β Hydroxylations. P. chrysosporium was more prone functionalize Steroids in the allylic position. Oxygen insertion at C-14 by M. plumbeus is reported for the first time. All three micro-organisms exhibited redox activity.
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Steroid Hydroxylation by Whetzelinia sclerotiorum, Phanerochaete chrysosporium and Mucor plumbeus.
Steroids, 2007Co-Authors: Andrew S Lamm, Avril R M Chen, William F Reynolds, Paul B ReeseAbstract:The fungi Whetzelinia sclerotiorum ATCC 18687, Phanerochaete chrysosporium ATCC 24725 and Mucor plumbeus ATCC 4740 were examined for their ability to perform Steroid biotransformations under single phase, pulse feed conditions. The Steroids 3beta-hydroxyandrost-5-en-17-one (dehydroepiandrosterone) (1), 17beta-hydroxyandrost-4-en-3-one (testosterone) (5), 3beta-hydroxypregn-5-en-20-one (pregnenolone) (3), pregn-4-ene-3,20-dione (progesterone) (9), 17alpha,21-dihydroxypregn-4-ene-3,11,20-trione (cortisone) (11), 17alpha,21-dihydroxypregna-1,4-diene-3,11,20-trione (prednisone) (14), and 3-hydroxyestra-1,3,5(10)-trien-17-one (estrone) (15) were fed to each fungus. The production of a number of novel metabolites is reported. Of the fungi investigated W. sclerotiorum performed the most interesting biotransformations and had a clear propensity for 2beta, 6beta/7beta and 15beta/16beta Hydroxylations. P. chrysosporium was more prone functionalize Steroids in the allylic position. Oxygen insertion at C-14 by M. plumbeus is reported for the first time. All three micro-organisms exhibited redox activity.
Andrew S Lamm - One of the best experts on this subject based on the ideXlab platform.
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Steroid Hydroxylation by whetzelinia sclerotiorum phanerochaete chrysosporium and mucor plumbeus
Steroids, 2007Co-Authors: Andrew S Lamm, Avril R M Chen, William F Reynolds, Paul B ReeseAbstract:Abstract The fungi Whetzelinia sclerotiorum ATCC 18687, Phanerochaete chrysosporium ATCC 24725 and Mucor plumbeus ATCC 4740 were examined for their ability to perform Steroid biotransformations under single phase, pulse feed conditions. The Steroids 3β-hydroxyandrost-5-en-17-one (dehydroepiandrosterone) (1), 17β-hydroxyandrost-4-en-3-one (testosterone) (5), 3β-hydroxypregn-5-en-20-one (pregnenolone) (3), pregn-4-ene-3,20-dione (progesterone) (9), 17α,21-dihydroxypregn-4-ene-3,11,20-trione (cortisone) (11), 17α,21-dihydroxypregna-1,4-diene-3,11,20-trione (prednisone) (14), and 3-hydroxyestra-1,3,5(10)-trien-17-one (estrone) (15) were fed to each fungus. The production of a number of novel metabolites is reported. Of the fungi investigated W. sclerotiorum performed the most interesting biotransformations and had a clear propensity for 2β, 6β/7β and 15β/16β Hydroxylations. P. chrysosporium was more prone functionalize Steroids in the allylic position. Oxygen insertion at C-14 by M. plumbeus is reported for the first time. All three micro-organisms exhibited redox activity.
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Steroid Hydroxylation by Whetzelinia sclerotiorum, Phanerochaete chrysosporium and Mucor plumbeus.
Steroids, 2007Co-Authors: Andrew S Lamm, Avril R M Chen, William F Reynolds, Paul B ReeseAbstract:The fungi Whetzelinia sclerotiorum ATCC 18687, Phanerochaete chrysosporium ATCC 24725 and Mucor plumbeus ATCC 4740 were examined for their ability to perform Steroid biotransformations under single phase, pulse feed conditions. The Steroids 3beta-hydroxyandrost-5-en-17-one (dehydroepiandrosterone) (1), 17beta-hydroxyandrost-4-en-3-one (testosterone) (5), 3beta-hydroxypregn-5-en-20-one (pregnenolone) (3), pregn-4-ene-3,20-dione (progesterone) (9), 17alpha,21-dihydroxypregn-4-ene-3,11,20-trione (cortisone) (11), 17alpha,21-dihydroxypregna-1,4-diene-3,11,20-trione (prednisone) (14), and 3-hydroxyestra-1,3,5(10)-trien-17-one (estrone) (15) were fed to each fungus. The production of a number of novel metabolites is reported. Of the fungi investigated W. sclerotiorum performed the most interesting biotransformations and had a clear propensity for 2beta, 6beta/7beta and 15beta/16beta Hydroxylations. P. chrysosporium was more prone functionalize Steroids in the allylic position. Oxygen insertion at C-14 by M. plumbeus is reported for the first time. All three micro-organisms exhibited redox activity.
Greg R. Harlow - One of the best experts on this subject based on the ideXlab platform.
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Use of chimeric enzymes and site-directed mutagenesis for identification of three key residues responsible for differences in Steroid Hydroxylation between canine cytochromes P-450 3A12 and 3A26.
Molecular pharmacology, 1999Co-Authors: David J. Fraser, Greg R. Harlow, James R. HalpertAbstract:Canine cytochromes P-450 3A12 and 3A26 differ by 22 out of 503 amino acid residues. Chimeric constructs and site-directed mutants were used to identify the residues responsible for the much higher rates of Steroid Hydroxylation by 3A12. Six initial 3A12/3A26 hybrids were generated using convenient restriction sites, and site-directed mutagenesis was used to restore full 3A12 activity to two of the hybrids. One pair of 3A12/3A26 chimeras indicated that the first four residue differences between 3A12 and 3A26 were at least partially responsible for the differences in progesterone Hydroxylation. Conversion in one of the hybrids of the Ile-187 residue found in 3A26 to the Thr in 3A12 conferred 3A12 levels of progesterone 6beta-hydroxylase activity. Analysis of another chimera identified key residues within an internal PstI fragment (codons 331-459) containing six amino acid residue differences. Subsequent site-directed mutagenesis of 3A26 residues Ser-368 and Val-369 to Pro and Ile, respectively, restored the rate of formation of 6beta-hydroxyprogesterone by the hybrid to that of 3A12. The simultaneous conversion of 3A26 residues 187, 368, and 369 to those of 3A12 conferred greater than a third of the progesterone 6beta-hydroxylase activity and all of the testosterone and androstenedione 6beta-hydroxylase activity of 3A12. Addition of the carboxyl terminal 44 3A12 residues to the 3A26 triple mutant doubled progesterone 6beta-hydroxylase activity. This is the first study to use catalytically distinct cytochromes P-450 3A from the same species in the elucidation of structure-function relationships.
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use of chimeric enzymes and site directed mutagenesis for identification of three key residues responsible for differences in Steroid Hydroxylation between canine cytochromes p 450 3a12 and 3a26
Molecular Pharmacology, 1999Co-Authors: David J. Fraser, Greg R. Harlow, James R. HalpertAbstract:Canine cytochromes P-450 3A12 and 3A26 differ by 22 out of 503 amino acid residues. Chimeric constructs and site-directed mutants were used to identify the residues responsible for the much higher rates of Steroid Hydroxylation by 3A12. Six initial 3A12/3A26 hybrids were generated using convenient restriction sites, and site-directed mutagenesis was used to restore full 3A12 activity to two of the hybrids. One pair of 3A12/3A26 chimeras indicated that the first four residue differences between 3A12 and 3A26 were at least partially responsible for the differences in progesterone Hydroxylation. Conversion in one of the hybrids of the Ile-187 residue found in 3A26 to the Thr in 3A12 conferred 3A12 levels of progesterone 6β-hydroxylase activity. Analysis of another chimera identified key residues within an internal Pst I fragment (codons 331–459) containing six amino acid residue differences. Subsequent site-directed mutagenesis of 3A26 residues Ser-368 and Val-369 to Pro and Ile, respectively, restored the rate of formation of 6β-hydroxyprogesterone by the hybrid to that of 3A12. The simultaneous conversion of 3A26 residues 187, 368, and 369 to those of 3A12 conferred greater than a third of the progesterone 6β-hydroxylase activity and all of the testosterone and androstenedione 6β-hydroxylase activity of 3A12. Addition of the carboxyl terminal 44 3A12 residues to the 3A26 triple mutant doubled progesterone 6β-hydroxylase activity. This is the first study to use catalytically distinct cytochromes P-450 3A from the same species in the elucidation of structure-function relationships.
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analysis of human cytochrome p450 3a4 cooperativity construction and characterization of a site directed mutant that displays hyperbolic Steroid Hydroxylation kinetics
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Greg R. Harlow, James R. HalpertAbstract:Cytochrome P450 3A4 is generally considered to be the most important human drug-metabolizing enzyme and is known to catalyze the oxidation of a number of substrates in a cooperative manner. An allosteric mechanism is usually invoked to explain the cooperativity. Based on a structure–activity study from another laboratory using various effector–substrate combinations and on our own studies using site-directed mutagenesis and computer modeling of P450 3A4, the most likely location of effector binding is in the active site along with the substrate. Our study was designed to test this hypothesis by replacing residues Leu-211 and Asp-214 with the larger Phe and Glu, respectively. These residues were predicted to constitute a portion of the effector binding site, and the substitutions were designed to mimic the action of the effector by reducing the size of the active site. The L211F/D214E double mutant displayed an increased rate of testosterone and progesterone 6β-Hydroxylation at low substrate concentrations and a decreased level of heterotropic stimulation elicited by α-naphthoflavone. Kinetic analyses of the double mutant revealed the absence of homotropic cooperativity with either Steroid substrate. At low substrate concentrations the Steroid 6β-hydroxylase activity of the wild-type enzyme was stimulated by a second Steroid, whereas L211F/D214E displayed simple substrate inhibition. To analyze L211F/D214E at a more mechanistic level, spectral binding studies were carried out. Testosterone binding by the wild-type enzyme displayed homotropic cooperativity, whereas substrate binding by L211F/D214E displayed hyperbolic behavior.