Theoretical Study On The Catalytic Mechanism Of Zinc-enzyme In The Cleavage Of C-N Bond

The reaction pathway of deformylation catalyzed by E coli. peptide deformylase (PDF) has been investigated by the density functional theory method of PBE1PBE on a small model and by a two-layer ONIOM method on a realistic protein model. The deformylation proceeds in sequential steps involving nucleophilic addition of metal coordinated water/hydroxide to the carbonyl carbon of the formyl peptide, proton transfer, and the cleavage of the C-N bond. The first step is the rate-determining step for the deformylation, which occurs through a penta-coordinated metal center. The estimated activation energies with the ONIOM method are about 23.0,15.0, and 14.9 kcal/mol for Zn-, Ni-, and Fe-PDFs, respectively. These calculated barriers are in close agreement with experimental observations. Our results demonstrate that the metal coordination geometry preference exerts significant influence on the catalytic activity of PDFs through affecting the activation of the carbonyl group of the substrate, the deprotonation of the metal-coordinated water, and the stabilization of the transition state. This preference for coordination geometry is mainly determined by the ligand environment and the intrinsic electronic structures of the metal center in the active site of PDFs.Based on the statistical results of crystal structures and theoretical calculation, the coordinated-ligands of zinc-peptidase mainly include two types:3His+H2O and 2His_Glu/Asp+H2O. All of zinc-peptidases need Glu or Asp to accept a proton from zinc-coordinated water molecule. The lewis acidity of zinc is influenced by the coordinated-ligands that can be neutral group or anionic group. As the coordinated-ligands of zinc are 3His+H2O, the extra hydrogen bond donor, such as Tyr, can stabilize the negative charge developed in the transition state and tetrahedral intermediate. But the extra hydrogen bond donor is not essential. As the coordinated-ligands of zinc are 2His_Glu/Asp+H2O, the zinc can’t stabilize the negative charge developed in the transition state and tetrahedral intermediate because the anionic group Gly/Asp reduces the lewis acidity of zinc. The extra hydrogen bond donors, such as Tyr, Arg and His, are needed to obtain the normal activity. In the Zn-PDF, the coordinated-ligands of zinc are 2His_Cys+H2O that has an anionic group. In this case, a hydrogen bond donor is essential to obtain the normal activity. However, there is no such residue found in the deformylase which can explain why Zn-PDF is almost inert.The reaction mechanism of Imidazolonepropionase (HutI) that catalyzes the hydrolysis of 4-imidazolone-5-propionic acid (IPA) to yield N-formimino-L-glutamic acid has been investigated using the two-layered ONIOM method. A novel mechanism has been proposed, which involves a key proton transfer from metal-coordinated water to Glu252, and then stabilized by the carbonyl oxygen of imidazolone ring of IPA via hydrogen bond, facilitating the nucleophilic attack on C4 of the imidazolone ring of IPA by Zn-coordinated hydroxide to form a tetrahedral intermediate. This occurs with a low barrier of about 3.0 kcal/mol. Then His272 mediates a proton transfer from the Zn-coordinated hydroxide to amino group of the tetrahedral intermediate, which is the rate-determining step of the whole process, with activation barrier of about 17.8 kcal/mol. Such calculated barrier is consistent with the activation barrier estimated from the experimental enzyme activity using transition state theory. The C4-N3 is then cleaved spontaneously to form the final product, and the more polar character of the product would facilitate its release from active site. This mechanism provides a suggestive model of understanding the reaction catalyzed by HutI, and also sheds light on the reaction mechanism of amidohydrolase superfamily.The reaction pathway of adenosine deamination catalyzed by adenosine deaminase (ADA) has been investigated by the two-layered ONIOM (PBE1PBE1: Amber) method on a realistic protein model. The deamination proceeds in four sequential steps involving the proton transfer from the zinc-coordinate water to the Glu217, nucleophilic addition of zinc-coordinated hydroxide to the adenine C6, proton transfer and cleavage of the C-N bond. The formation of tetrahedral intermediate through the nucleophilic addition in the second step is an exothermic step and the proton transfer from the tetrahedral intermediate to the amine group through a five-member ring transition state with the assistant of His238 is a rate-determine step.Our proposed catalytic mechanism for ADA is different from the previous mechanism, which is popularly accepted. In order to further testify our proposed mechanism, we test some kinetic data for ADA mutants and raise two crystals for special mutants(Y240E, Y240E/E217Q). These kinetic data and crystal structures are consistent with our proposed mechanism rather the previous mechanism.