The Crystal Structure of the H48Q Active Site Mutant of Human Group IIA Secreted Phospholipase A2 at 1.5 Å Resolution Provides an Insight into the Catalytic Mechanism

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• 作者：Suzanne H. Edwards ; Darren Thompson ; Sharon F. Baker ; Stephen P. Wood ; and David C. Wilton
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：December 31, 2002
• 年：2002
• 卷：41
• 期：52
• 页码：15468 - 15476
• 全文大小：270K
• 年卷期：v.41,no.52(December 31, 2002)
• ISSN：1520-4995

文摘

The human group IIA secreted PLA₂ is a 14 kDa calcium-dependent extracellular enzymethat has been characterized as an acute phase protein with important antimicrobial activity and has beenimplicated in signal transduction. The selective binding of this enzyme to the phospholipid substrate interfaceplays a crucial role in its physiological function. To study interfacial binding in the absence of catalysis,one strategy is to produce structurally intact but catalytically inactive mutants. The active site mutantsH48Q, H48N, and H48A had been prepared for the secreted PLA₂s from bovine pancreas and bee venomand retained minimal catalytic activity while the H48Q mutant showed the maximum structural integrity.Preparation of the mutant H48Q of the human group IIA enzyme unexpectedly produced an enzyme thatretained significant (2-4%) catalytic activity that was contrary to expectations in view of the acceptedcatalytic mechanism. In this paper it is established that the high residual activity of the H48Q mutant isgenuine, not due to contamination, and can be seen under a variety of assay conditions including assaysin the presence of Co²⁺ and Ni²⁺ in place of Ca²⁺. The crystallization of the H48Q mutant, yieldingdiffraction data to a resolution of 1.5 Å, allowed a comparison with the corresponding recombinant wild-type enzyme (N1A) that was also crystallized. This comparison revealed that all of the important featuresof the catalytic machinery were in place and the two structures were virtually superimposable. In particular,the catalytic calcium ion occupied an identical position in the active site of the two proteins, and thecatalytic water molecule (w6) was clearly resolved in the H48Q mutant. We propose that a variation ofthe calcium-coordinated oxyanion ("two water") mechanism involving hydrogen bonding rather than theanticipated full proton transfer to the histidine will best explain the ability of an active site glutamine toallow significant catalytic activity.