Selective probing of a NADPH site controlled light-induced enzymatic catalysis.

Achieving molecular recognition of NADPH binding sites is a compelling strategy to control many redox biologicalprocesses. The NADPH sites recognize the ubiquitous NADPH cofactor via highly conserved binding interactions,despite differences in the regulation of the hydride transfer in redox active proteins. We recently developed aphotoactive NADPH substitute, called nanotrigger NT synchronizing the initiation of enzymatic catalysis of theendothelial NO-synthase (eNOS) with a laser pulse. Spatial and temporal control of enzymatic activity by such adesigned light-driven activator would benefit from achieving molecular selectivity, i.e. activation of a singleNADPH-mediated enzyme.In this work, we probe the ability of NT to discriminate between two NADPH sites with light. The selected NADPHsitesbelongtodihydrofolatereductasedihydrofolatereductaseenzyme(DHFR)andendothelialNO-synthase(eNOS).Ultrafast kinetics showed that NT could not activate DHFR catalysis with a laser pulse in contrast with the observedtriggerofeNOScatalysisleadingtoNOformation.Homologymodelling,moleculardynamicssimulationsshowedthatNT discriminated between the two NADPH sites by different donor to acceptor distances and by local steric effectshinderinglightactivationofDHFRcatalysis.ThedatasuggestedthatthenarrowNADPHsiterequiredatightfitofthenanotriggeratasuitabledistance/angletotheelectronacceptorforaspecificactivationofthecatalysis.Theabilityofthe nanotrigger to activate eNOS combined with a low reactivity in unfavourable NADPH sites makes NT a highlypromising tool for targeting eNOS in endothelial cells with a laser pulse. Copyright 2009 John Wiley & Sons, Ltd.Supporting information may be found in the online version of this article.Keywords: docking; dynamic simulations; kinetics; molecular recognition; rational design; synchronization of enzymaticcatalysis