1h7k

From Proteopedia

Revision as of 14:26, 5 November 2007

FORMATION OF A TYROSYL RADICAL INTERMEDIATE IN PROTEUS MIRABILIS CATALASE BY DIRECTED MUTAGENESIS AND CONSEQUENCES FOR NUCLEOTIDE REACTIVITY

Overview

Proteus mirabilis catalase (PMC) belongs to the family of NADPH binding, catalases. The function of NADPH in these enzymes is still a matter of, debate. This study presents the effects of two independent phenylalanine, mutations (F194 and F215), located between NADPH and heme in the PMC, structure. The phenylalanines were replaced with tyrosines which we, predicted could carry radicals in a NADPH-heme electron transfer. The, X-ray crystal structures of the two mutants indicated that neither the, binding site of NADPH nor the immediate environment of the residues was, affected by the mutations. Measurements using H2O2 as a substrate, confirmed that the variants were as active as the native enzyme. With, equivalent amounts of peroxoacetic acid, wild-type PMC, F215Y PMC, and, beef liver catalase (BLC) formed a stable compound I, while the F194Y PMC, variant produced a compound I which was rapidly transformed into compound, II and a tyrosyl radical. EPR studies showed that this radical, generated, by the oxidation of Y194, was not related to the previously observed, radical in BLC, located on Y369. In the presence of excess NADPH, compound, I was reduced to a resting enzyme (k(obs) = 1.7 min(-1)) in a two-electron, process. This was independent of the enzyme's origin and did not require, any thus far identified tyrosyl radicals. Conversely, the presence of a, tyrosyl radical in F194Y PMC greatly enhanced the oxidation of reduced, beta-nicotinamide mononucleotide under a steady-state H2O2 flow with, observable compound II. This process could involve a one-electron, reduction of compound I via Y194.

About this Structure

1H7K is a Single protein structure of sequence from Proteus mirabilis with ACT, SO4 and HEM as ligands. Active as Catalase, with EC number 1.11.1.6 Structure known Active Site: AC1. Full crystallographic information is available from OCA.

Reference

Formation of a tyrosyl radical intermediate in Proteus mirabilis catalase by directed mutagenesis and consequences for nucleotide reactivity., Andreoletti P, Gambarelli S, Sainz G, Stojanoff V, White C, Desfonds G, Gagnon J, Gaillard J, Jouve HM, Biochemistry. 2001 Nov 13;40(45):13734-43. PMID:11695923

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