X-ray Absorption Spectroscopy And Crystallography Combination Of Zinc Metal The Protease Acutolysin-a And The Acutolysin-c Activity Center Structure And Function

Acutolysin-A and Acutolysin-C are purifed from Agkistrodon acutus venom and both of them belong to P-l-type snake venom metalloproteinases (SVMPs), with a molecular mass of about 23 kDa, which is a group of the adamalysin family of metzincins. The active-centers of metzincins are very conserved in structure. The evidence has shown that adamalysins and matrix metalloproteinases (MMPs) are involved in the same proteolytic mechanism. In this paper, we attempt to find out the structural base for the proteolytic activity and the catalytic mechanism of adamalysins as well as metzincins is further discussed.There is a significant improvement in the application of X-ray Absorption Spectroscopy (XAS) in biology. Particularly, the success development of the quantitative analysis method for X-ray Absorption Near-Edge Structure (XANES) leads to a promising future of application in more areas.A series of crystal structures of Acutolysin-A on different pH values have been collected and solved and the structures of previous data on pH 7.5 and pH 5.0 were also improved. The structures on all pH values exhibit a four-peptide carboxylate product in the P₁-P₄ substrate binding Site, as well as a three-peptides amine product present in the P₁'-P₄' Site of the structure on pH 9.0 which is close to the optimal pH value for catalytic activity. The structures of Acutolysin-A complexed with its products suggest the interaction states of the enzyme with its substrates and provide the direct support for the catalytic circle. Furthermore, all structures of Acutolysin-A we solved are the alternation states of the enzyme with either products or water molecules binding in the active pocket. The products combine with the enzyme, with an occupation of 52%-62% on different pH value and alternatively, the active pocket is occupied by the water molecules. The structures of active-center on different pH values reveal that the closer the pH value is to the optimal pH value for the proteolytic activity, the less distance there is between the catalytic-water-molecule (Wat1) with the carboxylate group OE2 atom of the conserved polarizing-residue Glu143. This result validates and improves further the previous structural explanation for the pH-dependent proteolytic mechanism.The XAS spectras of Acutolysin-C on different pH values have been measured by synchrotron-radiation-based XAS technique. The XANES spectra on pH 8.0 and pH 3.0 were fitted and two local zinc-center structure of Acutolysin-C in solution state have been returned using the MXAN procedure, a quantitative analysis code for XANES developed recently. In the structures on two pH values, we find that Wat1 moves far from the Glu143 residue from pH 8.0 to pH 3.0 and it is consistent with the research results of Acutolysin-A mentioned above. In addition, an auxiliary-catalytic water molecule Wat2 is observed near the zinc ion of free-form Acutolysin-C, which was also validated later by the crystallography result of 1.54 A PX data on pH 7.5. Comparing different structures by method of structure informatics, the conserved second residue behind the Met-turn (Met+2 site, most are Pro residue) is found to support the structure of Wat2, in all of adamalysins and MMPs. The main chain carbonyl group of Met+2 residue is always exposed to the 'auxiliary-catalytic region' where Wat2 is present (named totally 'Pro-Wat2' conformation). This feature is common among all metzincins which lack the auxiliary-catalytic Tyr residue in the active center. We believe that the 'Pro-Wat2' conformation has the same structure-conserved characteristic and intrinsic coherence in catalytic mechanism with the auxiliary-catalytic Tyr residue, in metzincin clan.