NTU Undergraduates' research April 2014 - Biosciences | Page 28

1

Nottingham Trent University, 2014

Comparison of 10-proline and 5-alanine mutants of the NADPH-dependant
wild type cytochrome P450 reductase and their effect on activation energy†
Rukhsaar Ali‡

Nottingham Trent University, The interdisciplinary biomedical research centre, Clifton Campus,
Cliftion Lane, Nottingham, NG11 8NS
Abstract: A combination of mutagenesis and spectrophotometer analysis has been used to study the
effect of mutants of human cytochrome P450 reductase (CPR) on the activation energy of the
reduction reaction. It has been demonstrated that the activation energy of the reaction is affected
by mutations taking place at an inter-domain flexible loop located 60 Å away. Substitution of a 7
amino acid long segment (T236-G237-E238-E239-S240-S241-I242) at this flexible loop by polyproline repeats of 10 residues results in a significant increase of 50% in the activation energy
required for the reduction of cytochrome c by NADPH dependant CPR, whereas a substitution of
poly-alanine repeats of 5 residues results in a slight decrease of activation energy of 4.16KJ/mol/K. It
has been proposed that there is a correlation between the length of amino acid chain at the flexible
loop of CPR and the overall activation energy required to initiate the reaction. The remarkable
increase in activation energy and reduced rate of reaction with the 10p mutant has been attributed
to an increase in relative chain stiffness produced by this mutation. Overall the findings of this paper
support earlier studies which demonstrate the effectiveness of the enzyme, and highlight how the
structure of the enzyme, particularly the flexible loop and the relative orientation of the FAD and
FMN-binding domains are suited exceptionally well to its function as a biological catalyst in essential
electron transfer processes.
Keywords: Cytrochrome P450 reductase; flexible region; mutants; conformational changes;
activation energy