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J Mol Biol. 2010 Nov 12;403(5):723-38. doi: 10.1016/j.jmb.2010.08.001. Epub 2010 Aug 12.

Dissecting the microscopic steps of the cyclophilin A enzymatic cycle on the biological HIV-1 capsid substrate by NMR.

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Department of Biochemistry and Howard Hughes Medical Institute, MS 009, Brandeis University, Waltham, MA 02454, USA.


Peptidyl-prolyl isomerases (PPIases) are emerging as key regulators of many diverse biological processes. Elucidating the role of PPIase activity in vivo has been challenging because mutagenesis of active-site residues not only reduces the catalytic activity of these enzymes but also dramatically affects substrate binding. Employing the cyclophilin A PPIase together with its biologically relevant and natively folded substrate, the N-terminal domain of the human immunodeficiency virus type 1 capsid (CA(N)) protein, we demonstrate here how to dissect residue-specific contributions to PPIase catalysis versus substrate binding utilizing NMR spectroscopy. Surprisingly, a number of cyclophilin A active-site mutants previously assumed to be strongly diminished in activity toward biological substrates based only on a peptide assay catalyze the human immunodeficiency virus capsid with wild-type activity but with a change in the rate-limiting step of the enzymatic cycle. The results illustrate that a quantitative analysis of catalysis using the biological substrates is critical when interpreting the effects of PPIase mutations in biological assays.

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