Substrate Recognition and Catalysis by UCH-L1

详细信息    查看全文

• 作者：Sarah J. Luchansky ; Peter T. Lansbury ; Jr. ; Ross L. Stein
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：December 12, 2006
• 年：2006
• 卷：45
• 期：49
• 页码：14717 - 14725
• 全文大小：192K
• 年卷期：v.45,no.49(December 12, 2006)
• ISSN：1520-4995

文摘

Deubiquitinating enzymes regulate essential cellular processes, and their dysregulation isimplicated in multiple disease states. Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) has garneredattention for its links with Parkinson's disease and cancer; however, the mechanism of action of thisenzyme in cells remains poorly understood. In order to advance our understanding of UCH-L1 function,we have been developing small molecule modulators of the enzyme for use as tools to probe its role incells. In support of these efforts, an investigation of the mechanism of UCH-L1 catalysis was previouslyreported. Here, we extend this mechanistic evaluation and examine substrate recognition by UCH-L1.We developed a panel of ubiquitin fusions to test the contribution of specific residues of ubiquitin tobinding and catalysis by the enzyme, and determined the activation parameters of selected variants togain additional mechanistic insight. Ubiquitin side chains critical for establishing the Michaelis complexand enabling catalysis were identified, and features of this complex that differ between UCH-L1 and ahomologue, UCH-L3, were revealed. These data provide dramatic examples of differences in substratespecificity between these enzymes. The implications of our experiments with UCH-L1 for selective inhibitordesign and the relationship to disease are discussed.