Kinetic and Structural Properties of Disulfide Engineered Phospholipase A2: Insight into the Role of Disulfide Bonding Patterns

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• 作者：Bao-Zhu Yu ; Ying H. Pan ; Marcel J. W. Janssen ; Brian J. Bahnson ; and Mahendra K. Jain
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：March 8, 2005
• 年：2005
• 卷：44
• 期：9
• 页码：3369 - 3379
• 全文大小：344K
• 年卷期：v.44,no.9(March 8, 2005)
• ISSN：1520-4995

文摘

The family of secreted 14 kDa phospholipase A₂ (PLA2) enzymes have a common motif forthe catalytic site but differ in their disulfide architecture. The functional significance of such structuralchanges has been analyzed by comparing the kinetic and spectroscopic properties of a series of disulfidemutants engineered into the sequence of pig pancreatic IB PLA2 to resemble the mammalian paraloguesof the PLA2 family [Janssen et al. (1999) Eur. J. Biochem. 261, 197-207, 1999]. We report a detailedcomparison of the functional parameters of pig iso-PLA2, as well as several of the human homologues,with these disulfide engineered mutants of pig IB PLA2. The crystal structure of the ligand free and theactive site inhibitor-MJ33 bound forms of PLA2 engineered to have the disulfide bonding pattern ofgroup-X (eng-X) are also reported and compared with the structure of group-IB and human group-XPLA2. The engineered mutants show noticeable functional differences that are rationalized in terms ofspectroscopic properties and the differences detected in the crystal structure of eng-X. A major differencebetween the eng-mutants is in the calcium binding to the enzyme in the aqueous phase, which also influencesthe binding of the active site directed ligands. We suggest that the disulfide architecture of the PLA2paralogues has a marginal influence on interface binding. In this comparison, the modest differencesobserved in the interfacial kinetics are attributed to the changes in the side chain residues. This in turninfluences the coupling of the catalytic cycle to the calcium binding and the interfacial binding event.