HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 276, Issue 33, 31459-31465, August 17, 2001

This Article Abstract Full Text (PDF) All Versions of this Article: 276/33/31459    most recent M103943200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nakayama, K. Articles by Pikuleva, I. A. Search for Related Content PubMed PubMed Citation Articles by Nakayama, K. Articles by Pikuleva, I. A. Social Bookmarking                      What's this?

Membrane Binding and Substrate Access Merge in Cytochrome P450 7A1, a Key Enzyme in Degradation of Cholesterol^*

Kazuo Nakayama, Andrei Puchkaev, and Irina A. Pikuleva^§

From the Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77555-1031

Received for publication, May 2, 2001, and in revised form, June 22, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To study membrane topology and mechanism for substrate specificity, we truncated residues 2-24 in microsomal cytochrome P450 7A1 (P450 7A1) and introduced conservative and nonconservative substitutions at positions 214-227. Heterologous expression in Escherichia coli was followed by investigation of the subcellular distribution of the mutant P450s and determination of the kinetic and substrate binding parameters for cholesterol. The results indicate that a hydrophobic region, comprising residues 214-227, forms a secondary site of attachment to the membrane in P450 7A1 in addition to the NH₂-terminal signal-anchor sequence. There are two groups of residues at this enzyme-membrane interface. The first are those whose mutation results in more cytosolic P450 (Val-214, His-225, and Met-226). The second group are those whose mutation leads to more membrane-bound P450 (Phe-215, Leu-218, Ile-224, and Phe-227). In addition, the V214A, V214L, V214T, F215A, F215L, F215Y, L218I, L218V, V219T, and M226A mutants showed a 5-12-fold increased K_m for cholesterol. The k_cat of the V214A, V214L, V219T, and M226A mutants was increased up to 1.8-fold, and that of the V214T, F215A, F215L, F215Y, L218I, and L218V mutants was decreased 3-10.5-fold. Based on analysis of these mutations we suggest that cholesterol enters P450 7A1 through the membrane, and Val-214, Phe-215, and Leu-218 are the residues located near the point of cholesterol entry. The results provide an understanding of both the P450 7A1-membrane interactions and the mechanism for substrate specificity.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The conversion of cholesterol into 7-hydroxycholesterol represents the first and rate-limiting step in overall bile acid biosynthesis, the major pathway for cholesterol elimination in mammals. This reaction takes place in the liver and is catalyzed by the microsomal enzyme cytochrome P450 7A1 (P450 7A1) (1, 2). Human P450 7A1 has been cloned (3) and expressed in Escherichia coli as a truncated (2-24) protein (4); however, little is known about the molecular basis for substrate specificity in this important P450.

Based on available crystal structures of eight different P450s that belong to distinct gene families (5-12), a paradigm is beginning to emerge: the mechanism for substrate specificity in the P450 superfamily is complex and determined not solely by residues located inside the enzyme active site (13-15). Unlike many enzymes, the substrate binding pocket of structurally characterized P450s does not form an open cleft at the molecular surface, but rather is buried inside the molecule. A channel connecting the protein exterior and the substrate binding site (substrate access channel) is seen in the structure of only two P450s, 102 and 51 (6, 12), and it is postulated that substrate entry in P450s is controlled by the opening motion of the substrate access channel (16). Although it is not clear how hydrophobic substrates reach the active site of soluble P450s, it has been proposed that in membrane-bound P450s the entrance of the substrate access channel is located within the membrane, thereby making it possible for lipophilic substrates to enter the enzyme directly from the lipid bilayer (10, 13). Peterson and colleagues (13) have also suggested that the hydrophobic substrate is recognized initially on the membrane-associated surface of the protein by a hydrophobic patch of amino acid residues adjacent to the substrate access channel. The substrate then enters the mouth of the access channel and partitions into the buried active site as a result of hydrophobic interactions with the residues lining the substrate access channel. These interactions determine the orientation of the substrate as it enters the P450 active site. Our recent studies of mitochondrial P450s 27A1 and 11A1, demonstrating that residues in the putative substrate access channel contribute to regioselectivity of hydroxylation in this subset of P450s (17), support Peterson's hypothesis.

Microsomal P450s have a highly hydrophobic segment of about 20 amino acid residues at the NH₂ terminus, which is believed to serve as a primary site of attachment to the endoplasmic reticulum (18). In addition, several microsomal P450s appear to have other regions that are inserted in and interact with the lipid bilayer because these P450s stay bound to the membrane after removal of the NH₂-terminal membrane anchor (4, 19-24). Site-directed mutagenesis and crystallographic studies of microsomal P450 2C5 identified topological elements that form the additional membrane binding sites in this enzyme (residues 30-45 before helix A, residues 60-69 following helix A, residues 376-379 and 201-210 that correspond to  strand 2-2, and the COOH-terminal end of the F-G loop, respectively) and suggested that association of P450s with the endoplasmic reticulum involves a monofacial hydrophobic surface of the protein formed by noncontiguous portions of the polypeptide chain (10). However, it is not established yet whether the same segments of the primary sequence are involved in the attachment to the membrane in different P450s.

In the present study, we used site-directed mutagenesis to examine the role of a hydrophobic region in P450 7A1 comprising residues 214-227 (the putative F-G loop and the adjacent helical segments). The results suggest that this region is a secondary membrane binding site in P450 7A1 and is also a region where cholesterol enters the enzyme active site. The work also provides experimental evidence that the mode of association of P450 7A1 with membrane is an important factor in regulating degradation of this physiologically important compound.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Construction of Expression Plasmids-- The cDNA for human P450 7A1 in pUC18 vector was obtained from Dr. D. Russell (University of Texas Southwestern Medical Center) and was used as a polymerase chain reaction template to modify the 5'- and 3'-portions of P450 7A1 cDNA. The 5'-primer for insertion of the full-length P450 7A1 into the expression vector was designed to introduce the NcoI site within the initiator codon ATG and to change the second codon ATG (Met) to GCT (Ala): 5'-CCCCCCATGGCTACCACATCTTTGATTTGGGGGATTGCT-3'. The 3'-primer was template-specific and contained a KpnI site downstream of the termination codon TGA: 5'-GAGCTCGGTACCTCACAAATGCTTGAATTTATATTTAAATTC-3'. The 5'-primer for insertion of the truncated (2-24) P450 7A1 into the expression vector contained the NcoI site within the initiator codon ATG, GCT as the second codon, and a sequence corresponding to the codons 25-32 of the full-length cDNA: 5'-CCCCCCATGGCTAGGAGAAGGCAAACGGGTGAACCA-3'. The 3'-primer was the same as for insertion of the full-length P450 7A1 into the expression vector. The polymerase chain reaction products were digested with NcoI and KpnI and then subcloned into the expression vector pTrc 99A (Amersham Pharmacia Biotech), digested previously with the same restriction enzymes. The correct sequence of the polymerase chain reaction-amplified fragments and the junction regions has been confirmed by DNA sequencing.

Site-directed Mutagenesis-- The QuickChange^TM site-directed mutagenesis kit (Stratagene) has been used to introduce point mutations into the cDNA encoding the truncated (2-24) P450 7A1 in pTrc 99A expression vector according to the instructions. Complementary mutagenic oligonucleotides used are shown in Table I. The correct generation of desired mutations and the absence of undesired mutations were confirmed by sequencing of the cDNA region from upstream of the EcoRI site to beyond the DraIII site. The EcoRI/DraIII fragments were then ligated into the EcoRI/DraIII-digested expression plasmid containing the wild type truncated P450 7A1. The subcloned fragments were then sequenced again to ensure the presence of the desired mutation and proper ligation.

Expression in E. coli-- Wild type and mutant P450s 7A1 were transformed into E. coli strain DH5F'IQ (Life Technologies, Inc.) and expressed as follows. An overnight culture was diluted 1:100 into TB medium containing 100 µg/ml ampicillin and grown at 37 °C with shaking at 250 rpm until the absorbance at 600 nm was 1.0. Isopropyl-1-thio--D-galactopyranoside, -aminolevulinic acid, and chloramphenicol were added to final concentrations of 1 mM, 0.5 mM and 1 µg/ml, respectively, and expression of P450 proceeded at 26 °C for 48 h with shaking at 190 rpm.

Subcellular Fractionation-- E. coli cells were harvested and suspended in 50% of the original culture volume in 10 mM potassium phosphate buffer (KP_i), pH 7.2, containing 20% glycerol. The cell suspension was incubated with 0.2 mg/ml lysozyme for 30 min on ice. Spheroplasts were pelleted at 3,000 × g for 20 min and then resuspended in 10 mM KP_i, pH 7.2, containing 20% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, 2 µg/ml aprotinin, and 1 µg/ml pepstatin. After sonication on ice using six 20-s pulses at 1-min intervals, cell debris was removed at 3,000 × g (20 min). The supernatant and pellet obtained after subsequent ultracentrifugation at 106,000 × g for 60 min were used as cytosolic and membrane fractions, respectively.

Quantification of P450 7A1 Subcellular Distribution-- Proteins in subcellular fractions were separated by SDS-polyacrylamide gel electrophoresis (5 µg of the total protein/lane) and transferred to nitrocellulose membrane. Western blot analysis was carried out using rabbit antiserum against P450 7A1 and an ECL detection system (Amersham Pharmacia Biotech) according to the manufacturer's instructions. X-ray films were scanned, and the immunoreactive signal was quantified using NIH Image 1.52 software. Only exposures lying within the film's linear range of sensitivity were used. Statistical analysis of differences between the wild type and the mutants was carried out using SigmaStat 2.0 software.

Partial Purification of Truncated P450 7A1-- Harvested cells were suspended in 10% of the original culture volume in 400 mM KP_i, pH 7.2, containing 1 M KCl and 20% glycerol, and incubated with 0.2 mg/ml lysozyme for 30 min on ice. After the addition of 0.5 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, 2 µg/ml aprotinin, and 1 µg/ml pepstatin, the cell suspension was sonicated on ice using six 20-s pulses at 1-min intervals and subjected to high speed centrifugation at 106,000 × g for 60 min. The supernatant obtained after ultracentrifugation was applied to an octyl-Sepharose column equilibrated with 100 mM KP_i, pH 7.2, 20% glycerol, and 0.3% sodium cholate. The column was washed with 5 column volumes of the equilibrating buffer, and P450 7A1 was eluted with the same buffer but containing 0.1% polyoxyethylene 10 lauryl ether. The fractions displaying a 450 nm CO difference spectrum were pooled, dialyzed against 10 mM KP_i, pH 7.2, and 20% glycerol and applied to a hydroxylapatite column equilibrated with the same buffer. The column was washed with 10 column volumes of the equilibrating buffer, and P450 7A1 was eluted with 400 mM KP_i, pH 7.2, 20% glycerol, and 0.15% sodium cholate. After dialysis against 40 mM KP_i, pH 7.2, and 20% glycerol, P450 7A1 was aliquoted and frozen at 70 °C. The specific heme content of P450 7A1 after this step was 5.1 nmol/mg of protein.

Enzyme Assay-- Cholesterol 7-hydroxylase activity of P450 7A1 wild type and mutants was assayed in 50 mM KP_i, pH 7.2, 0.05% Tween 20. The reaction mixture contained P450 7A1 (5-100 nM), 40 µg of L-dilauroylglyceryl-3-phosphocholine, 0.5-9 units of rat NADPH-cytochrome P450 reductase (1 unit reduces 1 µmol of cytochrome c/min), 2-200 µM cholesterol, and 120,000-240,000 cpm of [1,2-³H]cholesterol in a total volume of 1 ml. 1-10 mM cholesterol was added from a stock solution in 45% 2-hydroxypropyl--cyclodextrin. Reactions were initiated by the addition of 1 mM NADPH (final concentration) carried out at 37 °C for 1 min and terminated by adding 2 ml of CH₂Cl₂. After extraction, steroids were evaporated, dissolved in methanol, and separated by HPLC as described previously for the cholesterol 27-hydroxylase assay (25). The apparent K_m and V_max values were determined from Lineweaver-Burk plots. The k_cat value was obtained by dividing V_max by the molar enzyme concentration, which was measured by reduced a CO difference spectrum (26). In all studies, less than 15% of the total cholesterol was metabolized during enzymatic reaction, and product formation was linear with time and P450 7A1. Rat NADPH-cytochrome P450 reductase used in the enzymatic assay was expressed and purified as described (27). The expression plasmid pOR263 was kindly provided by Dr. F. Guengerich (Vanderbilt University).

Substrate Binding Assay-- Apparent binding constants (K_d) of P450 7A1 wild type and mutants for cholesterol were determined from the double reciprocal plot of a substrate-induced spectral change versus substrate concentration added. The assay was performed using 1-2 µM P450 at 12 °C in 1 ml of 50 mM KP_i, pH 7.2, containing 20% glycerol and 0.05% Tween 20. 1-10 mM cholesterol was added from a stock solution in 45% 2-hydroxypropyl--cyclodextrin. After each experiment, the P450 content was quantified to confirm that there was no denaturation during titration.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression of Full-length and Truncated (2-24) Human P450s 7A1 in E. coli-- Our approach was different from that reported earlier by Karam and Chiang (4). First, we utilized a different expression vector (pTrc 99A). Second, in the cDNA encoding the full-length enzyme, we replaced the second codon with an alanine codon (GCT) and left the third codon unchanged. Third, a different E. coli strain (DH5F'IQ) was used for protein expression. Fourth, optimal conditions for expression were established: temperature decreased to 26 °C, aeration speed increased to 190 rpm, and expression time increased to 48 h. Finally, during expression, we added chloramphenicol at the same time as isopropyl-1-thio--D-galactopyranoside and -aminolevulinic acid because we found that this antibiotic increases the level of expression of both full-length and truncated P450s 7A1 about 2-fold. Chloramphenicol was shown previously to enhance the expression of several different P450s (28). The resulting level of expression of the full-length and truncated P450s 7A1 was 50 and 520 nmol/liter of culture, respectively, which is higher than that reported by Li and Chiang for the full-length rat (29) and the truncated human enzymes (4).

Subcellular Distribution of the Full-length and Truncated (2-24) Human P450s 7A1 Expressed in E. coli-- Based on Western blot analysis, the full-length recombinant P450 7A1 is localized exclusively to the E. coli membrane fraction in both low (10 mM KP_i, 20% glycerol) and high (400 mM KP_i, 1 M KCl, 20% glycerol) ionic strength buffers, whereas about 40% of the truncated enzyme is found in the cytosol after subcellular fractionation in low ionic strength buffer. Twice as much (73%) truncated P450 7A1 is detected in the cytosol in high ionic strength buffer. Our results, which are in agreement with the literature data (4), confirm the membrane anchoring role of the NH₂-terminal residues 1-23 of the P450 and indicate the presence of additional (secondary) site(s) of attachment to the membrane. The solubilizing effect of high ionic strength implies peripheral association of the truncated form with E. coli membranes.

Identification of a Secondary Membrane Binding Site in P450 7A1-- Hydropathy plot analysis shows the presence of several hydrophobic regions in P450 7A1 in addition to the NH₂-terminal membrane anchor (Fig. 1). One of these regions (residues 214-227, Fig. 1) comprises the putative F-G loop, a topological element predicted to contribute to membrane association in microsomal P450s (13) and shown to contain residues involved in the interaction with membrane phospholipids in P450s 2B1 (30) and 2C5 (10). To begin to identify secondary membrane binding site(s) in P450 7A1, we used truncated (2-24) enzyme in which we initially substituted one-by-one alanine for all hydrophobic side chains located within the putative F-G loop and the adjacent helical segments. We also mutated His-225, which has a side chain that can be positively charged and could interact with negatively charged phospholipids. Mutant P450s were expressed in E. coli and their subcellular distribution in low ionic strength buffer investigated (Fig. 2). Replacement of a surface residue can increase the aggregation state of the P450 and result in appearance of the highly aggregated P450 in the membrane fraction, thus reflecting the limited solubility of the protein rather than specific membrane associations. Truncation significantly increased solubility of P450 7A1: the purified truncated protein remains soluble after the removal of detergent and can be concentrated until 30 mg/ml in the detergent-free buffer. Based on the dynamic light scattering, the truncated (2-24) P450 7A1 forms heptamers in detergent-free, low ionic strength buffer and remains in the supernatant under the conditions of subcellular fractionation (106,000 × g, 60 min). Substitution with alanine decreased the hydrophobicity of all, except histidine, wild type amino acid residues, making it unlikely to decrease the solubility of the mutant P450. As is described below, the H225A mutation increased a fraction of the P450 in the cytosol, which indicates that solubility of this mutant is not significantly affected. Thus, studies of subcellular distribution of the mutant P450s could be used as a method to assess the possible contribution of the residue in question to membrane-protein interactions, providing the solubility of the mutant P450 is not significantly decreased.

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Panel A, hydropathy plot of human P450 7A1 calculated using a hydrophobicity program of DNA Strider. Black bars indicate two hydrophobic regions investigated in the present study. Putative F and G helices in P450 7A1 were identified based on alignment with structurally characterized P450s 51, 107A1, 2C5, 101, and 102 (panel B) which was generated using the University of Wisconsin GCG Pileup program. This alignment was then refined manually, considering the structural alignments of P450s 101 and 102 (6) and 101 and 107A1 (8), the putative secondary structural elements in P450 7A1 (predicted using a consensus secondary structure prediction program, Web site http://pbil.ibcp.fr), and the sequence similarities. Original references for the P450 sequences can be found in the P450 superfamily summary (42). Boxes indicate  helices.

View larger version (41K): [in this window] [in a new window]   Fig. 2.   Effect of mutations on subcellular distribution of truncated (2-24) P450 7A1 in low ionic strength buffer (10 mM KPi, pH 7.4, 20% glycerol). Representative Western blots are shown on the left. M and C indicate the membrane fraction and cytosol, respectively. Subcellular fractionation was carried out as described under "Experimental Procedures." Fraction of P450 7A1 in the cytosol is expressed as a percentage of the total recovered in the membrane fraction and the cytosol. In all cases two independent expression experiments were carried out to isolate two sets of membrane fraction and cytosol. SDS-polyacrylamide gel electrophoresis was then carried out for each set independently, and one or two different gels were run independently for each set followed by Western blot analysis. The results represent the average of three to four different Western blot analyses ± S.D. Mean values that are significantly different (p < 0.05) from that of the truncated (2-24) wild type P450 7A1 are designated by an asterisk. The dashed line shows the percentage of the wild type enzyme in the cytosol. Volumes of the amino acids are taken from Ref. 43. Hydrophobicity of the amino acids is based on the normalized consensus hydrophobicity scale (44). Numbers in parentheses represent the volumes and the hydrophobicity of the wild type amino acid residue.

The V214A, H225A, and M226A replacements led to statistically significant increases in cytosolic P450 in contrast to alanine substitutions of Phe-215, Leu-218, Ile-224, and Phe-227, which resulted in up to a 1.5-fold increase of membrane-bound P450. Two mutants (V219A and L222A) showed no changes in subcellular distribution compared with that of the wild type truncated P450 7A1, indicating that Val-219 and Leu-222 are interior residues.

We also investigated the subcellular distribution of the mutants that were generated as part of the kinetic studies (described below) (Fig. 2). Replacement of Val-214 with Leu, which is bigger but of similar hydrophobicity, had little effect on P450 7A1 membrane binding properties. In contrast, substitution with the smaller and less hydrophobic Thr increased the amount of P450 in the cytosol, as observed for the alanine substitution. Thr is much more polar than Ala; however, the extent of retention of the V214T mutant in the membrane was the same as that of the V214A mutant, suggesting that it is the decreased size rather than the increased polarity of the side chain at position 214 which resulted in more cytosolic P450 7A1. Substitutions of Phe-215 with Leu, which is slightly smaller than Phe but twice as big as Ala, and with Tyr, which is structurally similar but much more polar than Phe, did not significantly affect the subcellular distribution of the mutant P450s. These data indicate that tighter membrane association of the F215A mutant is most probably caused by the reduction of the size of the hydrophobic side chain at position 215. Nonconservative and conservative replacements of Leu-218 with the smaller residues (the L218N and L218V mutations) led to more membrane-bound P450, although the substitutions had very different hydrophobicity, ranging from the same as that of the wild type to a very polar Asn. This is in contrast to the same size substitution with Ile resulting in no effect on subcellular distribution. A tighter binding of the L218N mutant compared with that of the L218A and L218V mutants could also be caused by the formation of the additional hydrogen bond(s) between the amide group of Asn and the phosphate moieties of the membrane. The V219T mutation, resulting in no alteration of the subcellular distribution, confirmed our prediction that the side chain of Val-219 is directed inside the protein molecule and not involved in the interaction with membrane.

Kinetic and Substrate Binding Properties of Mutant P450s-- Truncation of wild type P450 7A1 did not affect either K_m or k_cat, validating the use of the truncated form for our studies. Partial purification of truncated P450 7A1 resulted in a 2-fold reduction of the K_m and 3-fold reduction of the k_cat, suggesting that enzyme-membrane interactions or/and membrane phospholipids affect the P450 7A1 kinetic parameters. Initially we investigated nine alanine mutants that were generated for membrane topology studies. Based on kinetic properties, the mutants fall into four groups. The first group includes the V219A, L222A, and H225A mutants, the K_m and k_cat of which were only slightly different (up to 2.4-fold) from the kinetic parameters of the wild type P450. The data suggest that the mutated residues are not directly involved in cholesterol metabolism. The second group is formed by the I224A and F227A mutants, which had very low enzymatic activity, making it difficult to determine the kinetic parameters. Dramatic reduction of the enzymatic activity could be associated with alteration of P450-phospholipid interactions resulting from tighter binding of these mutants to the membrane. It should be noted that the extremely low enzymatic activity of the I224A and F227A mutants is not the result of structural perturbations around the heme caused by the mutations because the membrane fractions of these mutants showed a peak at 450 nm in the reduced CO difference spectrum, indicating the integrity of the P450 heme environment (26). The third group is represented by the F215A mutant. As with the previous group, the F215A mutant is also bound more tightly to the membrane than the wild type, indicating changes in membrane-enzyme interactions. To eliminate the possible effect of altered membrane-enzyme interactions, cytosol was used for determination of the kinetic parameters, which were compared with that of the partially purified enzyme. The F215A mutant exhibited a 5.4-fold increase in K_m and 4.5-fold decrease in k_cat, indicating that Phe-215 plays a role in cholesterol metabolism. Finally, the fourth group consists of the V214A, L218A, and M226A mutants, which have a 4.3-7.0-fold increase in K_m and insignificant (up to 2-fold) change in k_cat. Membrane fractions were used to determine the kinetic parameters of the V214A and M226A mutants, which are less tightly bound to the membrane, and cytosol was used to study the L218A mutant, which is bound more tightly to the membrane than the wild type.

To test that an increase of the K_m value of the V214A, F215A, and L218A mutants is not a result of microconformational changes introduced by the substitution and the altered enzyme-membrane interactions, we generated additional mutants with side chains of different sizes and hydrophobicity. Val-214 was replaced with a Leu and with Thr. Although the V214L and V214T mutations had different effects on subcellular distribution, and cytosol was used to determine the kinetic parameters of the V214L mutant, both substitutions increased the K_m, as did the alanine mutation, with the effect being more pronounced for a more polar replacement. Phe-215 was substituted with the structurally unrelated Leu, which has a similar size and hydrophobicity, and with structurally similar, but much more polar, Tyr. The K_m of the F215Y mutant was higher than that of the F215L and F215A mutants, indicating that both the size and polarity of the side chain at position 215 affect the K_m value. Leu-218 was replaced with structurally similar Ile and Val, and smaller and much more polar Asn. Conservative substitution with Ile did not affect the subcellular distribution; however, it increased the K_m more than 7-fold. The L218V mutation resulted in a tighter binding to a membrane and about 7-fold increase in the K_m value. Nonconservative L218N mutation leading to a further increase in the extent of membrane binding completely abolished enzyme activity. Thus, size and polarity of three surface hydrophobic residues at positions 214, 215, and 218 appear to be important factors that affect the K_m for cholesterol.

We also mutated Val-219, the side chain of which is directed inside the protein molecule, to a much more polar Thr. In contrast to alanine substitution, the mutation increased the K_m value about 9-fold, indicating that hydrophobicity of a residue at position 219 may aid in sequestering and binding of cholesterol to the buried active site. We hypothesize that Val-219 lines the interior of the substrate access channel.

The apparent dissociation constants (K_d values) for cholesterol were measured for the wild type truncated P450 7A1 and some of the mutants that exhibited increased K_m (Table II). The V214A, F215Y, and L218I mutations did not alter the K_d for cholesterol, suggesting that the affinity for the substrate is unchanged; however, these substitutions decreased the maximum amplitude of the substrate-induced spectral response (A_max) 3.3-10-fold.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The results obtained in this study indicate that a hydrophobic region within residues 214-227 is located on the surface of the P450 7A1 molecule and serves as a site of attachment to the membrane complementing the NH₂-terminal membrane anchor. Looser association of the V214A, H225A, and M226A mutants with the membrane indicates that either the replacements caused microconformational changes, leading to alteration of P450-membrane contacts, or that Val-214, His-225, and Met-226 are the surface residues that participate directly in the interactions with the lipid bilayer. Binding of P450s to membranes is most probably realized through multiple amino acid residues. A modest increase in cytosolic P450 (1.14-1.18-fold) argues against microconformational changes, in our opinion, and is more consistent with elimination of a contribution of a single amino acid residue. Moreover, Val-214, His-225, and Met-226, which are putatively located within the helical segments, were replaced by Ala, a residue that has a high helix propensity (31). Such replacement should not disrupt the helical structure of the region and consequently should not cause microconformational changes. An interesting finding was that smaller size F215A, L218A, L218N, L218V, I224A, and F227A substitutions resulted in a tighter binding to the membrane. This could imply that Phe-215, Leu-218, Ile-224, and Phe-227 are located on the surface of the enzyme and through spatial constraints control the depth of the P450 7A1 insertion in the membrane. Thus the strength of the membrane association of truncated P450 7A1 appears to be determined by the two types of residues: those that are directly involved in the interaction with membrane phospholipids and those that play a structural role and prevent deeper protein insertion in the membrane. The latter group of residues was identified because truncated enzyme was used to study the effect of mutations on subcellular distribution. Truncation led to about 40% of P450 7A1 in the cytosol, making it possible to monitor whether mutations resulted in more or less membrane-bound P450.

Another important finding was that the V214A, F215Y, and L218I mutants exhibited a 5-12-fold increase in the K_m value with an unchanged K_d value for cholesterol. K_m can be expressed as K_m = k₁/k₁ + k₂/k₁, where k₁/k₁ is a K_d, the dissociation constant of the Michaelis complex; k₁ is the rate of substrate release from enzyme active site; k₁ is the rate of substrate binding to enzyme active site; and k₂ is the rate of the product formation, also known as k_cat (32). K_m is therefore also a measure of enzyme affinity for its substrate, providing that k₂/k₁ is small compared with K_d; that is, k₂ < k₁ (32). The K_d value of the wild type truncated enzyme appeared to be very similar to the K_m value, indicating that in P450 7A1, binding and release of cholesterol occur much faster than k_cat. An increase in the K_m value of the V214A, F215Y, and L218I mutants was not associated with the decreased affinity for cholesterol, implying alteration of the on and off rates of cholesterol binding to the active site of these mutants. The V214A, F215Y, and L218I mutants also showed a 3.3-10-fold decrease of the amplitude of the substrate-induced spectral response, A_max. It is known that a spectral shift takes place when a water molecule is displaced by substrate from the coordination sphere of the heme iron (33). The change of the A_max points to either diminished amounts of cholesterol in the active site of the mutant enzymes and/or to changes in cholesterol orientation within the P450 active site. It is possible that because of mutation the substrate adopts an orientation in the active site which does not induce a spectral shift. Combined with the data on subcellular distribution, kinetic and substrate binding studies indicate that cholesterol comes to P450 7A1 through the membrane and that three surface residues at the membrane-protein interface, Val-214, Phe-215, and Leu-218, are important for cholesterol access to the P450 7A1 active site. We suggest that these residues are adjacent to the entry of the substrate access channel and that they participate in substrate recognition, which involves initial docking of cholesterol and determines the orientation of the substrate as it enters the substrate access channel and then the enzyme active site. Interaction of cholesterol with the surface hydrophobic residues may also trigger an opening motion of the substrate access channel, if it is in closed conformation in the substrate-free form of the enzyme. Even conservative substitutions of Val-214, Phe-215, and Leu-218 caused an increase in the K_m value, indicating that initial enzyme-substrate association is very specific, although not very strong.

Establishing that cholesterol enters P450 7A1 through the membrane also allows an explanation of how residues at the enzyme-membrane interface could contribute to the efficiency of catalysis. All three mutants (L218N, I224A, and F227A) which exhibited either no or significantly reduced enzymatic activity had altered enzyme-membrane interactions because they were bound more tightly to the membrane than the wild type enzyme. A dramatic decrease in enzymatic activity could be explained by the fact that much less substrate reached the active site of the mutant P450 compared with that of the wild type enzyme, and under subsaturating concentrations of cholesterol, less product was formed within the time of the assay. The results, thus, indicate that the mode of association of P450 7A1 with the membrane can also influence the efficiency of cholesterol degradation. Cholesterol has several possible fates within the hepatocytes. It can be incorporated into cell membranes, converted to cholesteryl esters that are packaged into very low density lipoprotein particles and secreted into sinusoids, catabolized to bile acids that are secreted into the bile, or be directly secreted into the bile (34). Mechanisms regulating the flux of cholesterol into these diverse pathways have yet to be delineated. We hypothesize that P4507A1-membrane interactions, which presumably depend on composition and content of membrane phospholipids, could be one of the factors that affects the metabolic fate of cholesterol.

A a methodology of how to investigate substrate recognition and access to P450 active site is being developed. Photoacoustic calorimetry (35), stop-flow techniques (33), and measurements of the substrate binding (36-38) have been utilized to study this phenomenon in structurally characterized, soluble bacterial P450s 101 and 102. Our work builds on identification of the surface residues in P450 whose tertiary structure is not yet known and comparison of the kinetic and substrate parameters of the wild type and mutant P450 7A1 with conservative and nonconservative substitutions of the surface residues. The results are concordant with both studies on membrane topology of P450s 2B1 and 2C5 and (10, 30) and several papers suggesting that the way in which P450s interact with membrane may be of importance for regulating substrate specificity (39-41). We propose that in eukaryotic P450s, which have strict substrate specificities and hydroxylate bulky hydrophobic substrates, recognition of the substrate takes place on the membrane-associated surface of the P450 molecule and serves as a mechanism to discriminate against the "wrong" substrates, allowing only the "right" substrate to enter the enzyme. However, further studies of other P450s are needed to validate this notion.

To summarize, the novelty of the present work is that it maps a secondary membrane binding site in P450 7A1, establishes that cholesterol accesses the P450 active site through the membrane, and that enzyme-membrane interactions are important for substrate specificity and effective catalysis; it also sheds light on a mechanism of regulation of cholesterol degradation in mammals and provides an example of methodology which identifies surface residues playing important roles in the metabolism of substrate. By providing further experimental verification of the concept that the surface of the P450 molecule participates in substrate recognition and extending this notion from bacterial P450 102 to a distantly related eukaryotic P450 7A1, this study also advances our understanding of the mechanism for substrate specificity in the P450 superfamily.

	ACKNOWLEDGEMENTS

Oligonucleotide synthesis and DNA sequencing were carried out by the Recombinant DNA Laboratory of the NIEHS Center and the Protein Chemistry Laboratory, respectively, at the University of Texas Medical Branch.

	FOOTNOTES

^* This work was supported by United States Public Health Service Grant GM62882-01, the John Sealy Memorial Endowment Fund, and Center Grant ES06676.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Permanent address: Institute of Bioorganic Chemistry, Byelorussian Academy of Sciences, Minsk 220141, Belarus.

^§ To whom correspondence should be addressed. Dept. of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1031. Tel.: 409-772-9657; Fax 409-772-9642; E-mail: irpikule@utmb.edu.

Published, JBC Papers in Press, June 22, 2001, DOI 10.1074/jbc.M103943200

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

1.	Myant, N. B., and Mitropoulos, K. A. (1977) J. Lipid Res. 18, 135-153
2.	Russell, D. W., and Setchell, K. D. R. (1992) Biochemistry 31, 4737-4749
3.	Karam, W. G., and Chiang, J. Y. L. (1992) Biochem. Biophys. Res. Commun. 185, 588-595
4.	Karam, W. G., and Chiang, J. Y. L. (1994) J. Lipid Res. 35, 1222-1231
5.	Poulos, T. L., Finzel, B. C., and Howard, A. J. (1987) J. Mol. Biol. 195, 687-700
6.	Ravichandran, K. G., Boddupalli, S. S., Hasemann, C. A., Peterson, J. A., and Deisenhofer, J. (1993) Science 261, 731-736
7.	Hasemann, C. A., Ravichandran, K. G., Peterson, J. A., and Deisenhofer, J. (1994) J. Mol. Biol. 236, 1169-1185
8.	Cupp-Vickery, J. R., and Poulos, T. L. (1995) Nat. Struct. Biol. 2, 144-153
9.	Park, S. Y., Shimizy, H., Adachi, S., Nakagawa, A., Tanaka, I., Nakahara, K., Shoun, H., Obayashi, E., Nakamura, H., Iizuka, T., and Shiro, Y. (1997) Nat. Struct. Biol. 4, 827-832
10.	Williams, P. A., Cosme, J., Sridhar, V., Johnson, E. F., and McRee, D. E. (2000) Mol. Cell 5, 121-131
11.	Yano, J. K., Koo, L. S., Schuller, D. J., Li, H., Ortiz de Montellano, P., and Poulos, T. (2000) J. Biol. Chem. 275, 31086-31092
12.	Podust, L. M., Poulos, T. L., and Waterman, M. R. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 3068-3073
13.	Graham-Lorence, S., Amarneh, B., White, R. E., Peterson, J. A., and Simpson, E. R. (1995) Protein Sci. 4, 1065-1080
14.	Graham, S., and Peterson, J. A. (1999) Arch. Biochem. Biophys. 369, 24-29
15.	Hoch, U., Falck, J. R., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 26952-26958
16.	Poulos, T. L., Finzel, B. C., and Howard, A. J. (1986) Biochemistry 25, 5314-5322
17.	Pikuleva, I. A., Puchkaev, A., and Björkhem, I. (2001) Biochemistry 40, 7621-7629
18.	Wachenfeldt, C., and Johnson, E. F. (1995) in Cytochrome P450: Structure, Mechanism, and Biochemistry (Ortiz de Montellano, P. R., ed), 2nd Ed. , pp. 183-244, Plenum Publishing Corp., New York
19.	Pernecky, S. J., Larson, J. R., Philpot, R. M., and Coon, M. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2651-26555
20.	Sagara, Y., Barnes, H. J., and Waterman, M. R. (1993) Arch. Biochem. Biophys. 304, 272-278
21.	Kempf, A. C., Zanger, U. M., and Meyer, U. A. (1995) Arch. Biochem. Biophys. 321, 277-288
22.	Dong, M.-S., Bell, L. C., Phillips, D. R., Blair, I. A., and Guengerich, F. P. (1996) Biochemistry 35, 10031-10040
23.	Wachenfeldt, C., Richardson, T. H., Cosme, J., and Johnson, E. F. (1997) Arch. Biochem. Biophys. 339, 107-114
24.	Cosme, J., and Johnson, E. F. (2000) J. Biol. Chem. 275, 2545-2553
25.	Pikuleva, I. A., Björkhem, I., and Waterman, M. R. (1997) Arch. Biochem. Biophys. 343, 123-130
26.	Omura, T., and Sato, R. (1964) J. Biol. Chem. 239, 2370-2378
27.	Hanna, I. H., Teiber, J. F., Kokones, K. L., and Hollenberg, P. F. (1998) Arch. Biochem. Biophys. 350, 324-332
28.	Kusano, K., Waterman, M. R., Sakaguchi, M., Omura, T., and Kagawa, N. (1999) Arch. Biochem. Biophys. 367, 129-136
29.	Li, Y. C., and Chiang, J. Y. (1991) J. Biol. Chem. 266, 19186-19191
30.	De Lemos-Chiarandini, C., Frey, A. B., Sabatini, D. D., and Kreibich, G. (1987) J. Cell Biol. 104, 209-219
31.	Pace, C. N., and Scholtz, J. M. (1998) Biophys. J. 75, 422-427
32.	Voet, D., and Voet, J. G. (1990) in Biochemistry (Stiefel, J., ed) , p. 337, John Wiley and Sons, Inc., New York
33.	Mueller, E. J., Loida, P. J., and Sligar, S. G. (1995) in Cytochrome P450: Structure, Mechanism, and Biochemistry (Ortiz de Montellano, P. R., ed), 2nd Ed. , pp. 183-244, Plenum Publishing Corp., New York
34.	Liscum, L., and Dahl, N. K. (1992) J. Lipid Res. 33, 1239-1254
35.	Wells, A. V., Li, P., Champion, P. M., Martinis, S. A., and Sligar, S. G. (1992) Biochemistry 31, 4384-4393
36.	Deprez, E., Gerber, N. C., Di Primo, C., Douzou, P., Sligar, S. G., and Hui bon Hoa, G. (1994) Biochemistry 33, 14464-14468
37.	Graham-Lorence, S. E., Truan, G., Peterson, J. A., Falck, J. R., Wei, S., Helvig, C., and Capdevila, J. H. (1997) J. Biol. Chem. 272, 1127-1135
38.	Oliver, C. F., Modi, S., Primrose, W. U., Lian, L. Y., and Roberts, G. C. K. (1997) Biochem. J. 327, 537-544
39.	Christou, M., Mitchell, M. J., Aoyama, T., Gelboin, H. V., Gonzalez, F. J., and Jefcoate, C. R. (1992) Biochemistry 31, 2835-2841
40.	Kominami, S., Itoh, Y., and Takemori, S. (1986) J. Biol. Chem. 261, 2077-2083
41.	Korzekwa, K. R., and Jones, J. P. (1993) Pharmacogenomics 3, 1-18
42.	Nelson, D. R., Koymans, L., Kamataki, T., Stegeman, J. J., Feyereisen, R., Waxman, D. J., Waterman, M. R., Gotoh, O., Coon, M. J., Estabrook, R. W., Gunsalus, I. C., and Nebert, D. W. (1996) Pharmacogenetics 6, 1-42
43.	Zamyatin, A. A. (1972) Prog. Biophys. Mol. Biol. 24, 107-123
44.	Eisenberg, D., Schwartz, E., Komarony, M., and Wall, R. (1984) J. Mol. Biol. 179, 125-142

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				I. A. Pikuleva CHOLESTEROL-METABOLIZING CYTOCHROMES P450 Drug Metab. Dispos., April 1, 2006; 34(4): 513 - 520. [Abstract] [Full Text] [PDF]

				N. Mast and I. A. Pikuleva A simple and rapid method to measure cholesterol binding to P450s and other proteins J. Lipid Res., July 1, 2005; 46(7): 1561 - 1568. [Abstract] [Full Text] [PDF]

				S. Kumar, E. E. Scott, H. Liu, and J. R. Halpert A Rational Approach to Re-engineer Cytochrome P450 2B1 Regioselectivity Based on the Crystal Structure of Cytochrome P450 2C5 J. Biol. Chem., May 2, 2003; 278(19): 17178 - 17184. [Abstract] [Full Text] [PDF]

				D. Murtazina, A. V. Puchkaev, C. H. Schein, N. Oezguen, W. Braun, A. Nanavati, and I. A. Pikuleva Membrane-Protein Interactions Contribute to Efficient 27-Hydroxylation of Cholesterol by Mitochondrial Cytochrome P450 27A1 J. Biol. Chem., September 27, 2002; 277(40): 37582 - 37589. [Abstract] [Full Text] [PDF]

				K. K. Khan, Y. Q. He, M. A. Correia, and J. R. Halpert Differential Oxidation of Mifepristone by Cytochromes P450 3A4 and 3A5: Selective Inactivation of P450 3A4 Drug Metab. Dispos., September 1, 2002; 30(9): 985 - 990. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 276/33/31459    most recent M103943200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nakayama, K. Articles by Pikuleva, I. A. Search for Related Content PubMed PubMed Citation Articles by Nakayama, K. Articles by Pikuleva, I. A. Social Bookmarking                      What's this?