THE DIVALENT-CATION IS OBLIGATORY FOR THE BINDING OF LIGANDS TO THE CATALYTIC SITE OF SECRETED PHOSPHOLIPASE-A(2)

被引:124
作者
YU, BZ
BERG, OG
JAIN, MK
机构
[1] UNIV DELAWARE, DEPT CHEM & BIOCHEM, NEWARK, DE 19716 USA
[2] UNIV UPPSALA, CTR BIOMED, DEPT MOLEC BIOL, S-75123 UPPSALA, SWEDEN
关键词
D O I
10.1021/bi00076a024
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The divalent cation requirement for partial reactions of the catalytic turnover cycle during interfacial catalysis by pig pancreatic phospholipase A2 (PLA2) is investigated. Results show that the specific role of calcium in all the events of the catalytic cycle at the active site is not shared by other divalent cations. Cations such as calcium, barium, and cadmium bind to the enzyme in the aqueous phase. The active-site-directed ligands (substrate, products, and transition-state mimics) do not bind to the enzyme in the absence of a divalent cation. The synergistic binding of such ligands to the active site of PLA2 bound to the interface is, however, observed only in the presence of isosteric ions like calcium and cadmium, but not with larger ions like strontium or barium. The equilibrium constants for ligands bound to the enzyme in the presence of calcium and cadmium are virtually the same. However, only calcium supports the catalytic turnover; the rate of hydrolysis in the presence of cadmium is less than 1% of that observed with calcium. The role of divalent ions on the interfacial catalytic turnover cycle of PLA2 is not only due to the cation-assisted binding of the substrate but also due to its participation in the chemical step. Other roles of divalent ions in the events of interfacial catalytic turnover are also identified. For example, the binding of the enzyme to the interface is apparently promoted because the divalent cation is required for the sequential step, i.e., the binding of the substrate to the active site of PLA2. Apparent activation of PLA2 also occurs by nonspecific effects related to the effect of cations on the organization of the substrate interface; for example, cations increase the apparent rate of hydrolysis by promoting the rate of substrate replenishment on the enzyme-containing particles by inducing the intervesicle exchange of the enzyme (Jain et al., 1986b) or by promoting fusion of vesicles (Jain et al., 1986a).
引用
收藏
页码:6485 / 6492
页数:8
相关论文
共 33 条
[1]   INTERFACIAL CATALYSIS BY PHOSPHOLIPASE-A2 - DETERMINATION OF THE INTERFACIAL KINETIC RATE CONSTANTS [J].
BERG, OG ;
YU, BZ ;
ROGERS, J ;
JAIN, MK .
BIOCHEMISTRY, 1991, 30 (29) :7283-7297
[2]   PHOSPHOLIPASE-A AND ITS ZYMOGEN FROM PORCINE PANCREAS .3. ACTION OF ENZYME ON SHORT-CHAIN LECITHINS [J].
DEHAAS, GH ;
BONSEN, PPM ;
PIETERSON, WA ;
VANDEENE.LL .
BIOCHIMICA ET BIOPHYSICA ACTA, 1971, 239 (02) :252-+
[3]   STRUCTURE OF BOVINE PANCREATIC PHOSPHOLIPASE-A2 AT 1.7A RESOLUTION [J].
DIJKSTRA, BW ;
KALK, KH ;
HOL, WGJ ;
DRENTH, J .
JOURNAL OF MOLECULAR BIOLOGY, 1981, 147 (01) :97-123
[4]   PHOSPHOLIPASE-A2 ENGINEERING - THE STRUCTURAL AND FUNCTIONAL ROLES OF AROMATICITY AND HYDROPHOBICITY IN THE CONSERVED PHENYLALANINE-22 AND PHENYLALANINE-106 AROMATIC SANDWICH [J].
DUPUREUR, CM ;
YU, BZ ;
MAMONE, JA ;
JAIN, MK ;
TSAI, MD .
BIOCHEMISTRY, 1992, 31 (43) :10576-10583
[5]   PHOSPHOLIPASE-A(2) ENGINEERING - STRUCTURAL AND FUNCTIONAL ROLES OF HIGHLY CONSERVED ACTIVE-SITE RESIDUES TYROSINE-52 AND TYROSINE-73 [J].
DUPUREUR, CM ;
YU, BZ ;
JAIN, MK ;
NOEL, JP ;
DENG, TL ;
LI, YS ;
BYEON, IJL ;
TSAI, MD .
BIOCHEMISTRY, 1992, 31 (28) :6402-6413
[6]   MODIFICATION OF CARBOXYLATE GROUPS IN BOVINE PANCREATIC PHOSPHOLIPASE-A2 - IDENTIFICATION OF ASPARTATE-49 AS CA2+-BINDING LIGAND [J].
FLEER, EAM ;
VERHEIJ, HM ;
DEHAAS, GH .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1981, 113 (02) :283-288
[7]  
Jain M K, 1992, Mediators Inflamm, V1, P85, DOI 10.1155/S0962935192000164
[8]   INTERFACIAL CATALYSIS BY PHOSPHOLIPASE-A2 - MONOMERIC ENZYME IS FULLY CATALYTICALLY ACTIVE AT THE BILAYER INTERFACE [J].
JAIN, MK ;
RANADIVE, G ;
YU, BZ ;
VERHEIJ, HM .
BIOCHEMISTRY, 1991, 30 (29) :7330-7340
[9]   INTERFACIAL CATALYSIS BY PHOSPHOLIPASE-A2 - DISSOCIATION-CONSTANTS FOR CALCIUM, SUBSTRATE, PRODUCTS, AND COMPETITIVE INHIBITORS [J].
JAIN, MK ;
YU, BZ ;
ROGERS, J ;
RANADIVE, GN ;
BERG, OG .
BIOCHEMISTRY, 1991, 30 (29) :7306-7317
[10]   ACTIVE-SITE-DIRECTED SPECIFIC COMPETITIVE INHIBITORS OF PHOSPHOLIPASE-A2 - NOVEL TRANSITION-STATE ANALOGS [J].
JAIN, MK ;
TAO, WJ ;
ROGERS, J ;
ARENSON, C ;
EIBL, H ;
YU, BZ .
BIOCHEMISTRY, 1991, 30 (42) :10256-10268