McGuire M, Carroll L J, Yankie L, Thrall S H, Dunaway-Mariano D, Herzberg O, Jayaram B, Haley B H
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742, USA.
Biochemistry. 1996 Jul 2;35(26):8544-52. doi: 10.1021/bi960275k.
Clostridium symbiosum pyruvate phosphate dikinase (PPDK) catalyzes the interconversion of adenosine 5'-triphosphate (ATP), orthophosphate (P(i)), and pyruvate with adenosine 5'-monophosphate (AMP), pyrophosphate (PP(i)), and phosphoenolpyruvate (PEP). The nucleotide binding site of this enzyme was labeled using the photoaffinity reagent [32P]-8-azidoadenosine 5'-triphosphate ([32P]-8-azidoATP). Subtilisin cleavage of the [alpha-32P]-8-azidoATP-photolabeled PPDK into domain-sized fragments, prior to SDS-PAGE analysis, allowed us to identify two sites of modification: one between residues 1 and 226 and the other between residues 227 and 334. Saturation of the ATP binding site with adenylyl imidodiphosphate afforded protection against photolabeling. Next, small peptide fragments of [gamma-32P]- 8-azidoATP-photolabeled PPDK were generated by treating the denatured protein with trypsin or alpha-chymotrypsin. A pair of overlapping radiolabeled peptide fragments were separated from the two digests, DMQDMEFTIEEGK (positions 318-330 in trypsin-treated PPDK) and RDMQDMEFTIEEGKL (positions 317-331 in alpha-chymotrypsin-treated PPDK), thus locating one of the positions of covalent modification. Next, catalysis by site-directed mutants generated by amino acid replacement of invariant residues of the PPDK N-terminal domain was tested. K163L, D168A, D170A, D175A, K177L, and G248I PPDK mutants retained substantial catalytic activity while G254I, R337L, and E323L PPDK mutants were inhibited. Comparison of the steady-state kinetic constants measured (at pH 6.8, 25 degrees C) for wild-type PPDK (kcat = 36 s-1, AMPK(m) = 7 microM, PP(i)K(m) = 70 microM, PEPK(m) = 27 microM) to those of R337L PPDK (kcat = 2 s-1, AMPK(m) = 85 microM, PP(i)K(m) = 3700 microM, PEPK(m) = 6 microM) and G254I PPDK (kcat = 0.1 s-1, AMPK(m) = 1300 microM, PP(i)K(m) = 1200 microM, PEPK(m) = 12 microM) indicated impaired catalysis of the nucleotide partial reaction (E.ATP.P(i) --> E-PP.AMP.P(i) --> E-P.AMP.PP(i) in these mutants. The single turnover reactions of [32P]PEP to [32P]E-P.pyruvate catalyzed by the PPDK mutants were shown to be comparable to those of wild-type PPDK. In contrast, the formation of [32P]E-PP/[32P]E-P in single turnover reactions of [beta-32P]ATP/P(i) was significantly inhibited. Finally, the location of the adenosine 5'-diphosphate binding site within the nucleotide binding domain of D-alanine-D-alanine ligase, a structural homologue of the PPDK N-terminal domain [Herzberg, O. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 2652-2657] indicates, by analogy, the location of the nucleotide binding site in PPDK. Residues G254, R337, and E323 as well as the site of photoaffinity labeling are located within this region.
共生梭菌丙酮酸磷酸二激酶（PPDK）催化三磷酸腺苷（ATP）、正磷酸盐（P(i)）和丙酮酸与一磷酸腺苷（AMP）、焦磷酸盐（PP(i)）和磷酸烯醇丙酮酸（PEP）之间的相互转化。使用光亲和试剂[32P]-8-叠氮基三磷酸腺苷（[32P]-8-azidoATP）标记该酶的核苷酸结合位点。在进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析之前，用枯草杆菌蛋白酶将[α-32P]-8-叠氮基ATP光标记的PPDK切割成结构域大小的片段，这使我们能够确定两个修饰位点：一个在第1位和第226位残基之间，另一个在第227位和第334位残基之间。用亚氨二磷酸腺苷使ATP结合位点饱和可提供对光标记的保护。接下来，通过用胰蛋白酶或α-胰凝乳蛋白酶处理变性蛋白，产生[γ-32P]-8-叠氮基ATP光标记的PPDK的小肽片段。从两种消化产物中分离出一对重叠的放射性标记肽片段，即DMQDMEFTIEEGK（胰蛋白酶处理的PPDK中第318 - 330位）和RDMQDMEFTIEEGKL（α-胰凝乳蛋白酶处理的PPDK中第317 - 331位），从而确定了共价修饰的一个位置。接下来，测试了通过氨基酸替换PPDK N端结构域不变残基产生的定点突变体的催化活性。K163L、D168A、D170A、D175A、K177L和G248I PPDK突变体保留了相当大的催化活性，而G254I、R337L和E323L PPDK突变体受到抑制。将在pH 6.8、25℃下测得的野生型PPDK（kcat = 36 s-1，AMPK(m) = 7 μM，PP(i)K(m) = 70 μM，PEPK(m) = 27 μM）的稳态动力学常数与R337L PPDK（kcat = 2 s-1，AMPK(m) = 85 μM，PP(i)K(m) = 3700 μM，PEPK(m) = 6 μM）和G254I PPDK（kcat = 0.1 s-1，AMPK(m) = [1300 μM，PP(i)K(m) = 1200 μM，PEPK(m) = 12 μM]）的进行比较，表明这些突变体中核苷酸部分反应（E.ATP.P(i) --> E-PP.AMP.P(i) --> E-P.AMP.PP(i)）的催化受损。PPDK突变体催化的[32P]PEP到[32P]E-P-丙酮酸的单周转反应显示与野生型PPDK相当。相比之下，[β-[32P]ATP/P(i)单周转反应中[32P]E-PP/[32P]E-P的形成受到显著抑制。最后，D-丙氨酸-D-丙氨酸连接酶（PPDK N端结构域的结构同源物[赫茨伯格，O.(1996)美国国家科学院院刊93, 2652 - 2657]）核苷酸结合结构域内二磷酸腺苷结合位点的位置通过类推表明了PPDK中核苷酸结合位点的位置。残基G254、R337和E323以及光亲和标记位点位于该区域内。
