Chemical modification of tyrosine‐38 in P‐Hydroxybenzoate hydroxylase from Pseudomonas fluorescens by 5′‐P‐fluorosulfonylbenzoyladenosine: A probe for the elucidation of the NADPH binding site?

Publisher Website
Google Scholar
Abstract
P‐Hydroxybenzoate Hydorxylase from Pseudomonas Fluorescens was covalently modified by the nucleotide analog 5′‐(P‐fluorosulfonylbenzoyl)‐adenosine in the presence of 20% dimethylsulfoxide, The inactivation reaction is pH‐dependent and does not obey pseudo‐first‐order kinetics, due to spontaneous hydrolysis of the reagent. The kinetic data further indicate that a weak, reversible enzyme‐inhibitor complex is an intermediate in the inactivation reaction and that only one amino acid residue is responsible for the loss of activityThe inactivation is strongly inhibited by NADPH and 2′, 5′ADP. Steady‐state kinetics and 2′,5′ADP bioaffinity chromatography of the modified enxzyme suggest that the enssential residue is not directly involved in NADPH binding.Sequence studies show that Tyr‐38 is the main residue protected form modicfication in the presence of NADPH. From crystallorgraphic studies it is known that the hydroxyl group of Tyr‐38 is 1.84 nm away form the active site. Model‐Building studies using computer graphics show that this distance can be accommodated when FSO2BzAdo binds in an extended comformation with the sulfonylbenzoyl protion in an orientaiton different from the nicotinamide ring of NADPH.

This publication has 39 references indexed in Scilit: