Activities of native and tyrosine-69 mutant phospholipases A2 on phospholipid analogs. A reevaluation of the minimal substrate requirements

Kuipers, Oscar P.; Dekker, Natashe Lemos; Verheij, Hubertus M.

doi:10.1021/bi00477a029

Cited by 32 publications

(33 citation statements)

References 36 publications

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“…piscivorus piscivorus Lys-49-PLA2 (group 11), it became clear that Lys-69 also binds to sn-3 phosphate (Scott et al, 1992). The mutation study for porcine pancreatic Asp-49-PLA2 showed that Tyr-69 plays a role for fixation of the phosphate group of the substrate and that replacement of Tyr-69 by lysine reduces enzymatic activity but retains the stereospecificity (Kuipers et al, 1990). Although such studies indicate that Lys-69 plays a role for interaction with the substrate, the present study showed that T. flavoviridis Asp-49-PLA2 trinitrophenylated at Lys-69 sustains 28% activity.…”

Section: Discussioncontrasting

confidence: 63%

Roles of lysine-69 in dimerization and activity ofTrimeresurus flavoviridis venom aspartate-49-phospholipase A2

Nakamura¹,

Nakai²,

Nakashima³

et al. 1996

J. Mol. Recognit.

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Trimeresurus flavoviridis (Habu snake) venom aspartate-49-phospholipase A2 (Asp-49-PLA2) was reacted at pH 9.0 with a 2-fold molar excess of 2,4,6-trinitrobenzenesulfonate in the absence of Ca2+ and two trinitrophenylated derivatives were isolated by HPLC. One was a derivative modified at Lys-11 and its activity was mostly retained. The other was a derivative modified at both Lys-11 and Lys-72 and its activity was 40% that of unmodified enzyme. Trinitrophenylation of Lys-72 appeared to bring about a conformational disorder at the lipid-water interface recognition site and thus a reduction of activity. When the enzyme was modified in the presence of Ca2+, activity decreased at a rate much faster than that in the absence of Ca2+ and Lys-69 came to be modified. These results suggested that conformational displacement of Asp-49-PLA2 of a local to global type occurs upon the binding of Ca2+. The derivative modified at Lys-69 had 28% activity and existed as a monomer. This supports a previous assumption that Lys-69 participates in dimerization of group II Asp-49-PLA2s [Brunie et al. (1985) J. Biol. Chem. 260, 9742-9749] and shows that dimerization is not necessarily essential for activity manifestation.

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Section: Discussioncontrasting

confidence: 63%

Roles of lysine-69 in dimerization and activity ofTrimeresurus flavoviridis venom aspartate-49-phospholipase A2

Nakamura¹,

Nakai²,

Nakashima³

et al. 1996

J. Mol. Recognit.

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“…Calcium is an obligatory cofactor for interfacial catalysis by PLA2, and this cation is coordinated to Asp-49, which is near His-48 (Verheij et al, 1980;Kuipers et al, 1990). Calcium is not required for the binding of PLA2 to DTPM vesicles (Jain et al, 1986b); however, as shown in Figure 2, calcium protects PLAZ from alkylation in a concentration-dependent manner.…”

Section: Principles and Experimental Strategy For Determiningmentioning

confidence: 99%

Interfacial catalysis by phospholipase A2: dissociation constants for calcium, substrate, products, and competitive inhibitors

Jain¹,

Bao²,

Rogers³

et al. 1991

Biochemistry

120

253

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Interpretation of the kinetics of interfacial catalysis in the scooting mode as developed in the first paper of this series [Berg et al. (1991) Biochemistry 30 (first paper of six in this issue)], was based on the binding equilibrium for a ligand to the catalytic site of phospholipase A2. In this paper, we describe direct methods to determine the value of the Michaelis-Menten constant (KMS) for the substrate, as well as the equilibrium dissociation constants for ligands (KL) such as inhibitors (KI), products (KP), calcium (KCa), and substrate analogues (KS) bound to the catalytic site of phospholipase A2 at the interface. The KL values were obtained by monitoring the susceptibility to alkylation of His-48 at the catalytic site of pig pancreatic PLA2 bound to micellar dispersions of the neutral diluent 2-hexadecyl-sn-glycero-3-phosphocholine. The binding of the enzyme to dispersions of this amphiphile alone had little effect on the inactivation rate. The half-time for inactivation of the enzyme bound to micelles of the neutral diluent depended not only on the nature of the alkylating agent but also on the structure and the mole fraction of other ligands at the interface. The KL values for ligands obtained from the protection studies were in excellent accord with those obtained by monitoring the activation or inhibition of hydrolysis of vesicles of 1,2-dimyristoyl-sn-glycerophosphomethanol. Since only calcium, competitive inhibitors, and substrate analogues protected phospholipase A2 from alkylation, this protocol offered an unequivocal method to discern active-site-directed inhibitors from nonspecific inhibitors of PLA2, such as local anesthetics, phenothiazines, mepacrine, peptides related to lipocortin, 7,7-dimethyleicosadienoic acid, quinacrine, and aristolochic acid, all of which did not have any effect on the kinetics of alkylation nor did they inhibit the catalysis in the scooting mode.

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“…Thus, the imidazole component of the Asp/His diad provides base catalysis of the direct attack of water at the carbonyl carbon of the substrate and the oxyanion hole contains a Ca 2 þ cation. The investigation of specificity and mechanism of phospholipase A 2 , particularly by crystallography [325,326] and protein engineering (e.g., [327,328]), and the development of kinetic methods for studies at lipid/water interfaces (surface dilution kinetics [329] and scooting kinetics [330] and references therein) are extensively reviewed [315]. Use of analog substrates with modified phospholipid head-groups and protein engineering experiments suggest that the interaction of the head-group with a tyrosine side-chain has a role in determining stereospecificity as well as catalytic activity [327].…”

Section: Lipasesmentioning

confidence: 99%

Substrate Recognition

Brocklehurst

Gul

Pickersgill

2009

Amino Acids, Peptides and Proteins in Organic Chemistry

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Specific molecular recognition processes by which molecules recognize and discriminate between closely related partners are involved in essentially all aspects of biological function, ranging in complexity from enzyme-inhibitor and enzymesubstrate systems to phenomena such as gene expression, the immune system, and synaptic transmission, and these are important considerations in drug design [1]. This chapter is concerned with general concepts exemplified by a selection of relatively well-defined enzyme-substrate and, by extension, enzyme-inhibitor systems. The original concept of recognition by complementarity in the enzymesubstrate adsorptive (Michaelis) complex was suggested by Emil Fischer in 1894 using his well-known lock-and-key analogy [2]. It is now generally accepted that this has now been superseded by complementarity in the reaction transition state, based on the ideas reported by J.B.S. Haldane [3] in 1930 and elaborated by Linus Pauling [4] in 1946. The catalytic potential of enzymes derives from stabilization in the transition state relative to any stabilization in the Michaelis complex. The latter is anticatalytic in that the free energy expended in stabilizing the adsorptive complex offsets the transition-state stabilization energy (e.g., [5]).Valuable chapters and books on enzyme catalysis and inhibition are available (e.g., [5][6][7][8][9][10][11]) and detailed mechanistic studies on many individual enzymes are described in Sinnot [12]. Macromolecular catalysis includes also catalysis by catalytic antibodies [13] and by synthetic polymers [14], including molecularly imprinted polymers [15]. The contributions of selections of various types of chemical catalysis such as acid-base, nucleophilic, and electrophilic catalysis [5,6,8] are almost always insufficient to account for the high catalytic efficiency of enzymes. A particularly important additional contribution is the entropic advantage that derives from the fact that enzyme catalysis occurs within an enzyme-substrate complex. The complex is essentially a single molecular species and the catalytic act involves one or more unimolecular steps during which there is no loss of translational or rotational entropy

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Activities of native and tyrosine-69 mutant phospholipases A2 on phospholipid analogs. A reevaluation of the minimal substrate requirements

Cited by 32 publications

References 36 publications

Roles of lysine-69 in dimerization and activity ofTrimeresurus flavoviridis venom aspartate-49-phospholipase A2

Roles of lysine-69 in dimerization and activity ofTrimeresurus flavoviridis venom aspartate-49-phospholipase A2

Interfacial catalysis by phospholipase A2: dissociation constants for calcium, substrate, products, and competitive inhibitors

Substrate Recognition

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