Veeraraghavan S, Nall B T
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
Biochemistry. 1994 Jan 25;33(3):687-92. doi: 10.1021/bi00169a009.
Structure-reactivity relationships of human peptidyl prolyl cis-trans isomerase (PPI) toward the two slow folding reactions of yeast iso-2 cytochrome c have been used to characterize the structure of folding intermediates in the vicinity of critical prolines. We propose that the relative catalytic efficiency of PPI for the protein substrate relative to a peptide substrate, (kcat/Km)rel, is a measure of structure in folding intermediates. The structural stability of slow-folding intermediates as detected by changes in (kcat/Km)rel was investigated using two structural perturbants: guanidine hydrochloride and site-directed mutagenesis. Neither of the two slow folding reactions for wild-type cytochrome c is catalyzed at low denaturant concentrations. However, both phases are catalyzed at moderate concentrations of guanidine hydrochloride. A mutation in cytochrome c enhances catalysis of the fluorescence-detected slow folding phase. For protein substrates destabilized by denaturants or mutation, we suggest that increases in (kcat/Km)rel result from a loosening of the substrate structure, providing better access of peptidyl prolyl isomerase to critical proline(s).
人类肽基脯氨酰顺反异构酶（PPI）对酵母同工酶-2细胞色素c的两个慢折叠反应的结构-反应性关系已被用于表征关键脯氨酸附近折叠中间体的结构。我们提出，PPI对蛋白质底物相对于肽底物的相对催化效率(kcat/Km)rel是折叠中间体结构的一种度量。使用两种结构扰动剂：盐酸胍和定点诱变，研究了通过(kcat/Km)rel变化检测到的慢折叠中间体的结构稳定性。在低变性剂浓度下，野生型细胞色素c的两个慢折叠反应均未被催化。然而，在中等浓度的盐酸胍下，两个阶段均被催化。细胞色素c中的一个突变增强了荧光检测到的慢折叠阶段的催化作用。对于因变性剂或突变而不稳定的蛋白质底物，我们认为(kcat/Km)rel的增加是由于底物结构的松弛，使肽基脯氨酰异构酶能够更好地接近关键脯氨酸。
