The Role of a Parasite-Specific Allosteric Site in the Distinctive Activation Behavior of Eimeria tenella cGMP-Dependent Protein Kinase

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• 作者：Scott P. Salowe ; Judyann Wiltsie ; Paul A. Liberator ; and Robert G. K. Donald
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：April 2, 2002
• 年：2002
• 卷：41
• 期：13
• 页码：4385 - 4391
• 全文大小：91K
• 年卷期：v.41,no.13(April 2, 2002)
• ISSN：1520-4995

文摘

A cGMP-dependent protein kinase (PKG) was recently identified as an anticoccidial targetfor the apicomplexan parasite Eimeria tenella [Gurnett, A., Liberator, P. A., Dulski, P., Salowe, S., Donald,R. G. K., Anderson, J., Wiltsie, J., Diaz, C., Harris, G., Chang, B., Darkin-Rattray, S. J., Nare, B., Crumley,T., Blum, P., Misura, A., Tamas, T., Sardana, M., Yuan, J., Biftu, T., and Schmatz, D. (2002) J. Biol.Chem. (in press)]. Unlike the PKGs of higher organisms that have two cGMP binding sites in theirregulatory domain, the PKG from Eimeria tenella (Et-PKG) contains three putative cGMP binding sitesand has distinctive activation properties, including a very large stimulation by cGMP (~1000-fold) withsignificant cooperativity (Hill coefficient of 1.7). During our investigation of Et-PKG activation, we foundthat 8-substituted cGMP analogues are weak partial activators. For example, 8-NBD-cGMP provides amaximal stimulation of activity of only 20-fold with little evident cooperativity, although cGMP cansynergize with the analogue to provide full activation. The results suggest that partial activation is aconsequence of restricted binding of 8-NBD-cGMP to a subset of cGMP sites in the enzyme. Site-directedmutagenesis of conserved arginine and glutamate residues in the parasite-specific third cGMP site confirmsthat this site is an important functional participant in the allosteric regulation of the kinase and that itexhibits very high selectivity against 8-NBD-cGMP. Since the results are consistent with full activationof Et-PKG requiring cyclic nucleotide binding in all three allosteric sites, one role for the additionalcGMP site may be to establish a stricter regulatory mechanism for the kinase activity than is present inthe PKGs of higher organisms containing only two allosteric sites.