Study On The Catalytic Mechanism And Dynamic Characteristics Of Nicotinamide N-methyltransferase

Nicotinamide N-methyltransferase（NNMT）is accountable to catalyze the N¹-methylation of nicotinamide（NCA）and makes a critical difference in liver detoxification and energy metabolism.Deviant expression of NNMT is connected with diverse diseases,containing various types of tumors,obesity,diabetes,and Parkinson’s disease.NNMT exists in the liver predominately but also expressed in some other organs.A rapid equilibrium ordered mechanism has been shown to be used by NNMT,with SAM binding first,followed by nicotinamide.From the crystal structure of human NNMT（PDB:3ROD）,three tryptophan residues,Trp97,Trp107,and Trp234 can be seen.Due to the low tryptophan content,the explanation of fluorescence data is not that complicated as there are no inter-tryptophan interactions.Besides,the crystal structure shows that Tyr20,as an active site residue,has a side chain hydroxyl group that interacts both to the nicotinamide ring and the carboxylate of S-adenosyl-L-homocysteine（SAH）.It’s shown that Tyr20 plays a vital role in NNMT function.Site-specific mutagenesis has been applied to generate Y20F and Y20G,which were used as probes for the sake of understanding the molecular mechanism and the dynamic properties of the NNMT catalytic reaction.An HPLC-MS/MS technology was used to measure the kinetic isotope effect of the reaction catalyzed by NNMT and its mutants.To better understand the catalytic behavior of enzymes,a model reaction was also introduced to compare with NNMT catalyzed system.Data shows that the catalytic efficiency is quite sensitive to the mutagenesis,showing around a 30-fold decrease for nicotinamide of Y20F(from 2488±309 M^-1S^-1for WT to 87±22 M^-1S^-1for Y20F),and an even more dramatic reduction（3.5×10⁴-fold reduction for nicotinamide）for Y20G.The smaller primary carbon KIE for NNMT WT compared with its mutants indicating that the transition state structure of NNMT WT is more asymmetric.Surprisingly,there is a significant trend that as the catalytic rate drops by 4 to 5 orders of magnitude,the secondary KIE changes from approximately 0.84 to 0.89（rising toward 0.941±0.003 for model reaction）.This means that NNMT WT has the tightest transition state structure and the highest catalytic efficiency.All in all,all of these observations are consistent with the notion that huge enzyme catalysis may result from transition state compression with shorter donor-accepter distance.Although extensive studies have concerned about the biological aspect,the detailed mechanism study of the enzyme function especially in the part of protein dynamics is lacking.Here,wild-type NNMT together with its Tyr20 mutants,and free tryptophan were carried out to explore the connection between protein dynamics and catalysis.Mono-tryptophan mutants were also designed to investigate their dynamic properties.An interesting phenomenon was observed:as the catalytic ability of the protein reduces,the fluorescence lifetime decreases with increasing protein flexibility.The data obtained indicate that compared with the Tyr20 mutants with lower activity,the catalytic movement of the substrate-binding domain at position 20 in wild-type NNMT prefers a less flexible structure.Studies on mono-tryptophan showed that Trp107 is more sensitive to the mutation at Tyr20 than Trp97.The Stokes shift results indicate that mutation on Tyr20 reduces the flexibility of the surrounding environment for protein tryptophan.Compared with the Tyr20 mutants,the smaller overall Stokes shift indicates a lower micro-polarity of the tryptophan microenvironment of“WT”protein.This project will help us to further understand the relative flexibility of protein structures,which cannot be too rigid or too flexible but strike a precise balance between achieving optimal catalytic function and optimal flexibility.