The first step in the synthesis of steroid hormones occurs in the mitochondria where cholesterol is converted to pregnenolone by cytochrome P450scc (CYP11A1). Cholesterol is insoluble in water and is supplied to the CYP11A1 directly from the inner mitochondrial membrane to which the enzyme is bound. The aim of this study was to characterise the interaction of bovine CYP11A1 with the phospholipid membrane. The effect of osmotic stress provided by glycerol on the spin-state, activity and degree of hydration of CYP11A1 was also investigated. Multiple sequence alignment of mitochondrial P450s revealed that there are 46 absolutely conserved residues, with the highest conservation in the heme-binding region at the C-terminal. The greatest variablility between subfamilies is in the regions believed to be involved in substrate binding (SRSs), as defined for the CYP2B family. The secondary structure prediction for CYP11A1 suggests that there is strong similarity in secondary structure to P450s of known structure. A model structure of CYP11A1 was built from primary sequence alignment to template P450 structures using the SwissModel automated server. From the model and other bioinformatic analyses, the distal face of the P450 which includes the A’ helix, F-G loop and beta sheet 1 regions, were predicted to interact with the membrane. Tryptic digests of CYP11A1 were performed with the aim of identifying membrane bound peptides that may be protected from protease activity. HPLC tryptic maps were similar in profile between soluble and vesicle-bound P450 which suggests that there is not a large region of CYP11A1 protected from protease digestion when the enzyme is attached to a membrane. Mass spectrometric analysis of peptides resulting from tryptic digestion revealed a number of peptides in the soluble digest that were not present in the digest of vesicle-bound P450. These peptides were located at the N-terminal and the J to J’ helix and interestingly, there was an absence of C-terminal peptides for both digests. This C-terminal peptide could be detected in digests of vesicle-bound P450 but not in digests of soluble P450 by tricine SDS polyacrylamide gel electrophoresis, Western transfer and N-terminal sequence analysis. Based upon the bioinfomatic and tryptic digestion data, a set of N- and C-terminal deletion mutants of CYP11A1 were expressed in E. coli and fractionated based on their association with the soluble or membrane fraction of the cells. The N-terminal deletion of the A’ helix resulted in an increase in the proportion of CYP11A1 in the soluble fraction while the C-terminal deletion did not alter membrane localisation. There are eight tryptophan residues in mature CYP11A1. The accessibility of these tryptophans to a water-soluble fluorescence quencher was determined for soluble and vesicle-bound enzyme. When CYP11A1 was associated with the vesicle membrane an average of 2 tryptophan residues were protected from quenching compared to soluble CYP11A1. This suggests that these tryptophan residues become buried within the membrane following association of CYP11A1 with the vesicles and are no longer accessible to quencher. The only free cysteine (C265S) of bovine CYP11A1 was removed by site directed mutagenesis and new cysteine residues introduced at selected sites based upon earlier results and the modelled CYP11A1 structure. The cysteine mutants were expressed, purified and labelled with the environmentally sensitive fluorescent probe, N-(7-nitrobenz-2-oxal-3-diazol-4-yl)ethylenediamine (NBD). There was an increase in the hydrophobicity of the NBD environment following the association of CYP11A1 with vesicles for the labeled mutants V212C and L219C. This indicates that these residues which are in the F-G loop, become localized to a more hydrophobic environment following membrane binding. Labeled cysteine residues introduced into the A’, B’ and G helices and β4-2 did not show major changes in hydrophobicity following membrane integration of CYP11A1. Osmotic stress of CYP11A1 induced by glycerol resulted in a low-spin spectral response and inhibition of activity. The change to low spin correlated with the dissociation of five or six water molecules from CYP11A1 and the inhibition of activity with cholesterol as substrate correlated with the dissociation of two molecules of water. In conclusion, this study shows that CYP11A1 is held to the membrane, at least in part, by the F-G loop region, and that the removal of water from the active site of CYP11A1 by osmotic stress causes a low spin spectral response and inhibition of activity.
| Identifer | oai:union.ndltd.org:ADTP/221033 |
| Date | January 2004 |
| Creators | Headlam, Madeleine Joyce |
| Publisher | University of Western Australia. School of Biomedical and Chemical Sciences |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Copyright Madeleine Joyce Headlam, http://www.itpo.uwa.edu.au/UWA-Computer-And-Software-Use-Regulations.html |
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