Nano-second Protein Dynamics In Catechol-O-Methyltransferase System: A Time-resolved Fluorescence Study

Human catechol-O-methyltransferase(COMT)is a key enzyme related to neurotransmitter metabolism and can catalyze a methyl transfer from S-adenosylmethionine(SAM)to catechol with an S_N2 ordered sequential reaction process.This process could effectively eliminate multiple signaling molecules(such as dopamine,epinephrine and norepinephrine),and is related to plenty of neurological diseases(anxiety,schizophrenia and Parkinson’s disease).But the physical origin of how this enzyme achieves high catalytic efficiency is still quite controversial with different models:the formation of near attack conformation,active site compaction,electrostatic preorganization and equatorial hydrogen bonding.And how protein dynamics contribute to its functions is still not clear.Thus,exploring the relationship between protein dynamics and protein properties is crucial to understand the catalytic mechanism of COMT.In this research,COMT W38in(Trp143Phe)and W38in/Y68A(Trp143Phe with Tyr68Ala)mutants were carried out and nanosecond resolved fluorescence spectroscopy was applied according to the intrinsic fluorescence property of tryptophan in it.When comparing the kinetic parameters,COMT W38in/Y68A presented a greater reduction compared with W38in.Then the comprehensive results of fluorescence decay lifetime,fluorescence quenching and Stokes shift in different solvents for the two COMT mutants showed that W38in/Y68A had a lower bimolecular quenching constant(k_q)and longer lifetime(τ)related with Stokes shift.These data implied the protein structure of mutant COMT W38in/Y68A with lower catalytic activity was more rigid than the“WT”-W38in,suggesting the importance of flexibility at residue 38 to maintain the optimal catalysis.And we proved that protein dynamics play an important role in protein functions.