Theoretical Studies Of The Catalytic Mechanisms Of Several Enzymes

Enzymes are biomacromolecules which catalyze almost all the intracellular chemical reactions. The metabolic processes catalyzed by enzymes are essential events for all the life. The high specificity of enzymes results from their unique three dimensional structures. Except a few catalytic DNA and RNA molecules, most enzymes are proteins. Along the development of technology and science, more and more enzymes are used in our daily life. Studies of the catalytic mechanisms or structures of enzymes at atomistic level can contribute to the understanding of the law and nature of life. Although some information such as the catalytic activity, crystal structures could be obtained via experimental methods, the details catalytic reaction mechanisms are not sure. The detailed information of enzymatic reactions can be obtained by using computational studies at atomic level. In this dissertation, several important enzymatic reactions have been studied by using combined quantum mechanics and molecular mechanics (QM/MM) method.The main contents as follow:(1) The catalytic mechanism of 7-carboxy-7-deazaguanine synthase (QueE).7-deazapurines-containing compounds are widely distributed in nature and include a series of structurally diverse nucleoside analogues that exhibit antineoplastic and antibiotic activities.7-deazapurine moieties are also found in the hypermodified tRNA nucleosides. The biosynthesis of 7-deazapurines involves four enzymes GCHI, QueC, QueD and QueE. Among,7-carboxy-7-deazaguanine synthase (QueE) is a SAM radical enzyme, which catalyzes the unprecedented rearrangement of 6-carboxy-5,6,7,8-tetrahydropterin (CPH4) into 7-carboxy-7-deazaguanine (CDG). Although QueE belongs to the SAM superfamily, it exhibits clear structural difference to the common SAM enzyme. Besides, QueE also shows a clear dependence on metal ions, and it is considered as a new feature for an AdoMet radical enzyme. For example, in the assays of QueE from B. multivorans, Mg2+ enhances the activity by three-fold, but Na+ does not accelerate the QueE activity. QueE is the first member of the SAM radical superfamily that requires a metal ion to assist its catalytic reaction. The complex and heterocyclic rearrangement of the carbon skeleton follows a radical-mediated mechanism. Up to now, there are two possible mechanisms for QueE, i.e. the reaction may undergo either generation of an imine intermediate, or yielding a bridged azacyclopropylcarbinyl intermediate. In 2014, the crystal structures of QueE from B. multivorans were successfully determined, in which all the cofactors were reported, including the intact substrate and the product. Based on the X-ray crystal structures of QueE in complex with the intact substrate, the catalytic mechanism of QueE from B. multivorans has been studied by using combined quantum mechanics and molecular mechanics (QM/MM) method. Our calculations reveal that the key ring contraction step involves a bridged intermediate rather than a ring-opening intermediate. The abstraction of pro-R C7 proton by the residue Glul 16 to aromatize the product is mediated by an active site water molecule. Compared with that of the QueE-Mg2+, the energy barriers of the QueE-Na+ complex show clearly increase. During the catalytic cycle, the Mg2+ and Na+ not only act as Lewis acids but also fix the substrate in its reactive conformation. The different coordination of Mg2+ and Na+ with the substrate is the main reason to lead the different activities of QueE-Mg2+ and QueE-Na+ complexes. Our results may provide useful information for further understanding the catalytic mechanism of QueE.(2) The catalytic mechanism of (R)-hydroxynitrile lyases (AtHNL).Hydroxynitrile lyases (HNLs) catalyze the conversion of chiral cyanohydrins to hydrocyanic acid (HCN) and aldehyde or ketone. The product HCN is a good weapon for defense against microbial and herbivores attack in cyanogenic plants, and can be used as a nitrogen source in the biosynthesis of asparagine. Most important, HNL can catalyze the asymmetric synthesis of cyanohydrins using the non-natural chiral substrate HCN and ketone as materials. The enantiomerically enriched cyanohydrin is a valuable intermediate in pharmaceuticals, agrochemicals, and chemicals, which are difficult to synthesize through the classical chemical methods. Hydroxynitrile lyase from Arabidopsis thaliana (AtHNL) is the first R-selective HNL enzyme containing an α/β-hydrolases fold. The discovery of AtHNL broke the accepted rule that only the S-selective hydroxynitrile lyases of the HNLs family contain an α/β-hydrolases fold. Like other α/β-hydrolases, the AtHNL catalytic reaction involves a Ser-His-Asp catalytic triad, and the important roles of these three residues have been demonstrated by site-directed mutagenesis. Although AtHNL has been identified for several years, the detailed reaction mechanism of AtHNL is still not clear. There are two possible mechanisms for AtHNL, one is suggested that the Ser residue usually acts as a nucleophile, the alternative is a general acid/base mechanism. In this article, the detailed catalytic mechanism of AtHNL was theoretically studied by using QM/MM approach based on the recently obtained crystal structure in 2012. During the calculations, two computational models were constructed, and two possible reaction pathways were considered. Our calculations reveal that the catalytic reaction of AtHNL involves a general acid/base mechanism. The free energy barriers of Path A and B are in good agreement with the experimental value, both Path A and B are suggested to be possible. Our calculations for the mechanism of AtHNL at atomistic level can provide useful help for the improvement of biocatalysts.(3) The catalytic mechanism of methylornithine synthase (Py1B).Pyrrolysine is the 22nd amino acid encoded by the natural genetic code. It increases the protein structure diversity. It is necessary for the conversion of methylamines to methane in all of the known pathways. Experimental evidences have confirmed that the carbon and nitrogen atoms of pyrrolysine are all derived from lysine, and Py1B is necessary for the biosynthesis of pyrrolysine. Methylomithine synthase (Py1B) belongs to the family of radical SAM enzymes which converts (2S)-lysine to (2R,3R)-3-methylornithine in a radical mechanism. In the catalytic reaction of Py1B, the S chiral centre of L-lysine is inverted to the R chiral centre of (2R,3R)-3-methylomithine. The lysine mutase reaction catalyzed by P1yB follows a fragmentation-recombination mechanism. It is the first proposed mechanism of mutase reaction for a SAM-dependent radical enzyme to catalyze the rearrangement of carbon backbone. In this paper, the mechanism of lysine mutase reaction catalyzed by Py1B has been studied by using quantum mechanics/molecular mechanics (QM/MM) approach. The calculations reveal that the PylB-catalyzed reaction involves seven elementary reaction steps. Both the hemolytic cleavage of Ca-Cβ bond of lysine and the ligation of glycyl radical with aminobutene are possible rate limiting, corresponding to the calculated energy barriers of 23.0 and 24.1 kcal/mol, respectively. The intramolecular rotation of a fragment (aminobutene) can well explain the stereochemistry of the final product. Our calculations agree well with the experimental results and can provide useful information for the further study of the radical enzyme.(4) The catalytic mechanism of cyclohexane-1,2-dione hydrolase (CDH).As the biologically active derivative of vitamin B1, thiamin diphosphate (ThDP) is a critical cofactor in numerous enzymes, and plays important roles in several biochemical reactions. ThDP is an important compound for maintain normal functions of digestive system, nervous system and heart. Cyclohexane-1,2-dione hydrolase (CDH) belongs to the ThDP family which is discovered in 2011. It is necessary for the biodegradation pathway of a-diketones of alicyclic ring compound. CDH catalyzes the conversion of cyclohexane-1,2-dione (CDO) to 6-oxohexanoate. It is the first a-ketolase known to date and the first ThDP-dependent enzyme so far that breaks a carbon-carbon bond ring of alicyclic compound. Although CDH has been experimentally investigated, the detailed catalytic mechanism of CDH is still not fully understood yet. In this paper, the detailed catalytic mechanism of CDH has been studied by using quantum mechanics/molecular mechanics (QM/MM) approach. Based on the protonation states of CDO, three computational models were constructed. In model A, the calculated catalytic reaction involves five elementary steps, and the cleavage of Cl (CDO)-C2(ThDP) bond is the rate limiting step with the energy barrier of 19.9 kcal/mol. For model B and B’, only model B can lead to the conversion of CDO to 6-oxohexanoate, and the corresponding reaction contains three elementary steps. Based on our comparison, it is concluded that the C-C bond cleavage is greatly facilitated by the deprotonation of CDO. Theoretical study on the catalytic mechanism of CDH can help our understanding of the catalytic properties of CDH and other ThDP-dependent enzymes.(5) The catalytic mechanism of human AMSH-LP domain deubiquitinating enzymes (DUBs).Ubiquitin is a post-translational modifier which regulates a wide variety of biological processes. It exists in all tissues of eukaryotic organisms and can attach to the lysine residues of the substrate protein by an isopeptide bond or to the amino group of the N-terminus of protein by a peptide bond. Similar to other post-translational modification, ubiquitylation is also a reversible process that can be controlled and regulated through deubiquitination process catalyzed by deubiquitinating enzymes (DUBs). DUBs catalyze the hydrolysis of peptide (isopeptide) bond between ubiquitin molecules, and remove the covalently linked ubiquitin molecules from the target substrates. The human AMSH-LP DUB domain specifically cleaves the isopeptide bonds of Lys63-linked polyubiquitin chains. Lys 63-linked ubiquitin chains play proteasome-independent roles in a variety of intracellular events, such as DNA repair, inflammatory signalling and ribosomal protein synthesis. AMSH is zinc metalloproteases which require a Zn2+ for catalysis. In this article, the catalytic mechanism of AMSH-LP has been studied by using combined quantum mechanics and molecular mechanics method. Two possible hydrolysis process (Path 1 and Path 2) were considered. Our calculations reveal that the activation of Zn2+-coordinated water molecule is the essential step for the hydrolysis of isopeptide bond. The activated hydroxyl firstly attacks the carbonyl group of Gly76, and then the amino group of Lys63 is protonated. According to our calculations, Zn2+ plays an important role in the catalytic reaction. Besides acting as Lewis acid, Zn2+ also influences the reaction by regulating its ligated environment.