Miller W T, Kaiser E T
Laboratory of Bioorganic Chemistry and Biochemistry, Rockefeller University, New York, NY 10021.
Proc Natl Acad Sci U S A. 1988 Aug;85(15):5429-33. doi: 10.1073/pnas.85.15.5429.
A peptide-based photoaffinity label for the catalytic subunit of the cAMP-dependent protein kinase was prepared from the amino acid p-benzoyl-L-phenylalanine [L-Phe(pBz)]. By using solid-phase peptide synthesis methodology, DL-Phe(pBz) was incorporated into the cAMP-dependent protein kinase substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly in place of the phosphorylatable serine. The diastereomeric peptides were separated by reverse-phase HPLC. The peptide substrate analog containing L-Phe(pBz) had a Ki of approximately 110 microM at pH 7.5. When photolyzed at 350 nm in the presence of the enzyme, this peptide caused time- and concentration-dependent inactivation. Radioactive acetylated L-Phe(pBz) peptide was used to establish the binding stoichiometry of peptide to enzyme; these results, together with protection experiments, showed the photoaffinity labeling to be specific (approximately 1:1). To identify the residues that were modified on the catalytic subunit, the photoinactivated enzyme was cleaved with CNBr and V8 protease (Staphylococcus aureus). The resulting peptide fragments were purified by HPLC and were sequenced; these experiments identified the modified residues as Gly-125 and Met-127. This region of the cAMP-dependent protein kinase catalytic subunit contains many residues that are conserved in serine- and tyrosine-protein kinases.
一种基于肽的环磷酸腺苷（cAMP）依赖性蛋白激酶催化亚基的光亲和标记物由氨基酸对苯甲酰基-L-苯丙氨酸[L-Phe(pBz)]制备而成。通过使用固相肽合成方法，将DL-Phe(pBz)掺入cAMP依赖性蛋白激酶底物Leu-Arg-Arg-Ala-Ser-Leu-Gly中，取代可磷酸化的丝氨酸。通过反相高效液相色谱法分离非对映异构体肽。含L-Phe(pBz)的肽底物类似物在pH 7.5时的解离常数（Ki）约为110微摩尔。当在酶存在下于350纳米处进行光解时，该肽导致时间和浓度依赖性失活。放射性乙酰化L-Phe(pBz)肽用于确定肽与酶的结合化学计量；这些结果与保护实验一起表明光亲和标记具有特异性（约为1:1）。为了鉴定催化亚基上被修饰的残基，用溴化氰（CNBr）和V8蛋白酶（金黄色葡萄球菌）切割光失活的酶。通过高效液相色谱法纯化所得的肽片段并进行测序；这些实验确定被修饰的残基为Gly-125和Met-127。cAMP依赖性蛋白激酶催化亚基的这一区域包含许多在丝氨酸和酪氨酸蛋白激酶中保守的残基。
