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A Single Residue With Dramatic Impact On Substrate‐ Induced Spin‐State Change In P450BM‐3
Author(s) -
Haines Donovan C.,
Hegde Amita,
Chen Baozhi,
Tomchick Diana,
Zhao Weiqiang,
Bondlela Muralidhar,
Humphreys John M.,
Mullin David A.,
Machius Mischa,
Peterson Julian A.
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.lb125
Subject(s) - chemistry , substrate (aquarium) , enzyme , conformational change , cytochrome p450 , residue (chemistry) , mutant , serine , active site , stereochemistry , heme , catalytic cycle , biophysics , biochemistry , biology , ecology , gene
Cytochrome P450 enzymes are important in Phase I xenobiotic metabolism, hormone metabolism, and the synthesis of numerous natural products. The bacterial enzyme P450BM‐3 has long served as a convenient model of P450 structure and function. As with many other P450s, P450BM‐3 undergoes a change in the spin‐state of the heme iron upon substrate binding. This change from the resting six‐coordinate low‐spin state to the five‐coordinate highspin state may help conserve electrons in the absence of oxidizable substrate, as the low‐spin state is harder to reduce. We have found that mutation of an active site alanine (A328) to valine produces an enzyme with greatly improved substrate binding, spin‐state conversion, and turnover rate. Mutation of the same residue to serine, however, results in greatly impaired spin‐state conversion and impaired substrate binding. Even more intriguing, the effect of the A328S mutant on V max depends on the type of substrate examine, with fatty acid oxidation being highly impaired but N‐acyl amino acid oxidation rate being enhanced ~60%. These results give us new insights into how protein conformational change couples substrate binding to spin‐state change. This research was supported in part by research grants GM43479 and GM50858 from the NIH (JAP) and X‐011 from the Robert A. Welch Foundation (DCH).