Pro Region C-Terminus:Protease Active Site Interactions Are Critical in Catalyzing the Folding of -Lytic Protease
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- 作者：Reuben J. Peters ; Andrew K. Shiau ; Julie L. Sohl ; D. Eric Anderson ; Gale Tang ; Joy L. Silen ; and David A. Agard
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：September 1, 1998
- 年：1998
- 卷：37
- 期：35
- 页码：12058 - 12067
- 全文大小：175K
- 年卷期：v.37,no.35(September 1, 1998)
- ISSN：1520-4995
文摘
-Lytic protease is encoded with a large (166 amino acid) N-terminal pro region that is requiredtransiently both in vivo and in vitro for the correct folding of the protease domain [Silen, J. L., andAgard, D. A. (1989) Nature 341, 462-464; Baker, D., et al. (1992) Nature 356, 263-265]. The proregion also acts as a potent inhibitor of the mature enzyme [Baker, D., et al. (1992) Proteins: Struct.,Funct., Genet. 12, 339-344]. This inhibition is mediated through direct steric occlusion of the activesite by the C-terminal residues of the pro region [Sohl, J. L., et al. (1997) Biochemistry 36, 3894-3904].Through mutagenesis and structure-function analyses we have begun to investigate the mechanism bywhich the pro region acts as a single turnover catalyst to facilitate folding of the mature protease. Ofcentral interest has been mapping the interface between the pro region and the protease and identifyinginteractions critical for stabilizing the rate-limiting folding transition state. Progressive C-terminal deletionsof the pro region were found to have drastic effects on the rate at which the pro region folds the proteasebut surprisingly little effect on inhibition of protease activity. The observed kinetic data strongly supporta model in which the detailed interactions between the pro region C-terminus and the protease areremarkably similar to those of known substrate/inhibitor complexes. Further, mutation of two proteaseresidues near the active site have significant effects on stabilization of the folding transition state (kcat) orin binding to the folding intermediate (KM). Our results suggest a model for the -lytic protease proregion-mediated folding reaction that may be generally applicable to other pro region-dependent foldingreactions.