Peptidyl Prolyl cis/trans-Isomerases: Comparative Reactivities of Cyclophilins, FK506-Binding Proteins, and Parvulins with Fluorinated Oligopeptide and Protein Substrates
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- 作者：Ralph Golbik ; Chao Yu ; Elisabeth Weyher-Stingl ; Robert Huber ; Luis Moroder ; Nediljko Budisa ; and Cordelia Schiene-Fischer
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：December 13, 2005
- 年：2005
- 卷：44
- 期：49
- 页码：16026 - 16034
- 全文大小：131K
- 年卷期：v.44,no.49(December 13, 2005)
- ISSN：1520-4995
文摘
Peptidyl prolyl cis/trans-isomerases catalyze the cis-trans isomerization of prolyl bonds inoligopeptides and various folding states of proteins. The proline residue in PPIase substrates at the P1'subsite, which follows the isomerizing peptide bond, appears to be the common recognition element forall subfamilies of this enzyme class. The molecular principles that govern substrate specificity at the P1'subsite were analyzed using 4-fluoroproline-containing tetrapeptide 4-nitroanilides and barstar Cys40Ala/Cys82Ala/Pro27Ala/Pro48
4-fluoroproline quadruple variants. Generally, PPIase catalysis demonstratedstereospecificity for monofluoro substitutions at the 4-position of the pyrrolidine ring. However, thereplacement of hydrogens with fluoro atoms did not impair productive interactions for the majority ofPPIase-substrate complexes. Comparison of specificity constants for oligopeptide and protein substratesrevealed striking differences in the 4-fluoroproline substituent effects between members of the PPIasefamilies. Introduction of 4(R)-fluoroproline resulted in an oligopeptide substrate completely resistant tocatalytic effects of FKBP-like PPIases. By contrast, the 4(R)-fluoroproline barstar variant demonstratedonly slightly reduced or even better catalytic susceptibility when compared to the parent barstar Cys40Ala/Cys82Ala/Pro27Ala/Pro48 substrate. On the other hand, Suc-Ala-Ser-4(S)-FPro-Phe-pNA exhibits adiscriminating specificity toward the prototypic parvulin, the Escherichia coli Par10. The E. coli triggerfactor, in the extreme, catalyzes Cys40Ala/Cys82Ala/Pro27Ala/4-F2Pro48 with a more than 20-fold higherefficiency when compared to the proline-containing congener. These findings support the combined subsiteconcept for PPIase catalysis in which the positioning of a substrate in the active cleft must activate a stillunknown number of remote subsites in the transition state of the reaction. The number of critical subsiteswas shown to vary between the PPIase families.
4-fluoroproline quadruple variants. Generally, PPIase catalysis demonstratedstereospecificity for monofluoro substitutions at the 4-position of the pyrrolidine ring. However, thereplacement of hydrogens with fluoro atoms did not impair productive interactions for the majority ofPPIase-substrate complexes. Comparison of specificity constants for oligopeptide and protein substratesrevealed striking differences in the 4-fluoroproline substituent effects between members of the PPIasefamilies. Introduction of 4(R)-fluoroproline resulted in an oligopeptide substrate completely resistant tocatalytic effects of FKBP-like PPIases. By contrast, the 4(R)-fluoroproline barstar variant demonstratedonly slightly reduced or even better catalytic susceptibility when compared to the parent barstar Cys40Ala/Cys82Ala/Pro27Ala/Pro48 substrate. On the other hand, Suc-Ala-Ser-4(S)-FPro-Phe-pNA exhibits adiscriminating specificity toward the prototypic parvulin, the Escherichia coli Par10. The E. coli triggerfactor, in the extreme, catalyzes Cys40Ala/Cys82Ala/Pro27Ala/4-F2Pro48 with a more than 20-fold higherefficiency when compared to the proline-containing congener. These findings support the combined subsiteconcept for PPIase catalysis in which the positioning of a substrate in the active cleft must activate a stillunknown number of remote subsites in the transition state of the reaction. The number of critical subsiteswas shown to vary between the PPIase families.