α-胰凝乳蛋白酶共价磷酸化衍生物的31P核磁共振

31P NMR of covalent phosphorylated derivatives of alpha-chymotrypsin.

作者信息

Gorenstein D G, Shah D, Chen R, Kallick D

机构信息

Department of Chemistry, Purdue University, West Lafayette, Indiana 47907.

出版信息

Biochemistry. 1989 Mar 7;28(5):2050-8. doi: 10.1021/bi00431a013.

DOI:10.1021/bi00431a013

PMID:2719945

Abstract

The structures of various covalent phosphorylated derivatives of alpha-chymotrypsin (alpha-CT) have been studied by 31P NMR spectroscopy. Diisopropylphosphoryl-alpha-chymotrypsin (alpha-DIPCT) shows a single 31P signal at ca. 0.0 ppm (pH 4). At low pH, the 31P NMR spectrum of alpha-DIPCT gradually changed with the appearance of one or two additional peaks. The ratio of the peaks varied with pH, time, and concentration. One of these two new downfield peaks (both at ca. 2.0 ppm) has been previously identified by Markley and co-workers (Markley, 1979; Porubcan et al., 1979) and van der Drift et al. (1985) as an aged monoisopropylphosphoryl-alpha-chymotrypsin (alpha-MIPCT) and is confirmed by our studies. A new additional downfield signal, separate from the alpha-MIPCT signal, is attributed to a dimer of the phosphorylated alpha-DIPCT. Phosphorylation of the enzyme with diphenyl chlorophosphate yields a monophenylphosphoryl-alpha-chymotrypsin (alpha-MPPCT) that also showed a single 31P signal at -2.1 ppm (pH 7). However, the spectrum did not change as a function of pH, incubation time, or concentration. Comparison of the 31P chemical shifts of the native and denatured phosphorylated derivatives of alpha-chymotrypsin suggests changes in the conformation about the P-O ester bonds are at least partially responsible for the various 31P chemical shift differences.

摘要

已通过³¹P核磁共振光谱研究了α-胰凝乳蛋白酶（α-CT）各种共价磷酸化衍生物的结构。二异丙基磷酰基-α-胰凝乳蛋白酶（α-DIPCT）在约0.0 ppm（pH 4）处显示单个³¹P信号。在低pH下，α-DIPCT的³¹P核磁共振光谱随着一个或两个额外峰的出现而逐渐变化。峰的比例随pH、时间和浓度而变化。这两个新的低场峰之一（均在约2.0 ppm处）先前已被马克利及其同事（马克利，1979年；波鲁布坎等人，1979年）以及范德德里夫特等人（1985年）鉴定为老化的单异丙基磷酰基-α-胰凝乳蛋白酶（α-MIPCT），并得到我们研究的证实。一个与α-MIPCT信号分开的新的额外低场信号归因于磷酸化的α-DIPCT的二聚体。用二苯基氯磷酸酯对该酶进行磷酸化产生单苯基磷酰基-α-胰凝乳蛋白酶（α-MPPCT），其在-2.1 ppm（pH 7）处也显示单个³¹P信号。然而，该光谱并未随pH、孵育时间或浓度而变化。α-胰凝乳蛋白酶天然和变性的磷酸化衍生物的³¹P化学位移比较表明，围绕P-O酯键的构象变化至少部分导致了各种³¹P化学位移差异。