Publication

Activities of Native and Tyrosine-69 Mutant Phospholipases A2 on Phospholipid Analogues. A Reevaluation of the Minimal Substrate Requirements

Kuipers, O. P., Dekker, N., Verheij, H. M. & Haas, G. H. D., 1990, In : Biochemistry. 29, 25, 9 p.

Research output: Contribution to journalArticleAcademic

APA

Kuipers, O. P., Dekker, N., Verheij, H. M., & Haas, G. H. D. (1990). Activities of Native and Tyrosine-69 Mutant Phospholipases A2 on Phospholipid Analogues. A Reevaluation of the Minimal Substrate Requirements. Biochemistry, 29(25). https://doi.org/10.1021/bi00477a029

Author

Kuipers, Oscar P. ; Dekker, Nicolaas ; Verheij, Hubertus M. ; Haas, Gerard H. de. / Activities of Native and Tyrosine-69 Mutant Phospholipases A2 on Phospholipid Analogues. A Reevaluation of the Minimal Substrate Requirements. In: Biochemistry. 1990 ; Vol. 29, No. 25.

Harvard

Kuipers, OP, Dekker, N, Verheij, HM & Haas, GHD 1990, 'Activities of Native and Tyrosine-69 Mutant Phospholipases A2 on Phospholipid Analogues. A Reevaluation of the Minimal Substrate Requirements', Biochemistry, vol. 29, no. 25. https://doi.org/10.1021/bi00477a029

Standard

Activities of Native and Tyrosine-69 Mutant Phospholipases A2 on Phospholipid Analogues. A Reevaluation of the Minimal Substrate Requirements. / Kuipers, Oscar P.; Dekker, Nicolaas; Verheij, Hubertus M.; Haas, Gerard H. de.

In: Biochemistry, Vol. 29, No. 25, 1990.

Research output: Contribution to journalArticleAcademic

Vancouver

Kuipers OP, Dekker N, Verheij HM, Haas GHD. Activities of Native and Tyrosine-69 Mutant Phospholipases A2 on Phospholipid Analogues. A Reevaluation of the Minimal Substrate Requirements. Biochemistry. 1990;29(25). https://doi.org/10.1021/bi00477a029


BibTeX

@article{944771b34fe74a6e842dae8937ea686a,
title = "Activities of Native and Tyrosine-69 Mutant Phospholipases A2 on Phospholipid Analogues. A Reevaluation of the Minimal Substrate Requirements",
abstract = "The role of Tyr-69 of porcine pancreatic phospholipase A2 in substrate binding was studied with the help of proteins modified by site-directed mutagenesis and phospholipid analogues with a changed head-group geometry. Two mutants were used containing Phe and Lys, respectively, at position 69. Modifications in the phospholipids included introduction of a sulfur at the phosphorus (thionophospholipids), removal of the negative charge at phosphorus (phosphatidic acid dimethyl ester), and reduction (phosphonolipids) or extension (diacylbutanetriol choline phosphate) of the distance between the phosphorus and the acyl ester bond. Replacement of Tyr-69 by Lys reduces enzymatic activity, but the mutant enzyme retains both the stereospecificity and positional specificity of native phospholipase A2. The Phe-69 mutant not only hydrolyzes the Rp isomer of thionophospholipids more efficiently than the wild-type enzyme, but the Sp thiono isomer is hydrolyzed too, although at a low (~4{\%}) rate. Phosphonolipids are hydrolyzed by native phospholipase A2 about 7 times more slowly than natural phospholipids, with retention of positional specificity and a (partial) loss of stereospecificity. The dimethyl ester of phosphatidic acid is degraded efficiently in a calcium-dependent and positional-specific way by native phospholipase A2 and by the mutants, indicating that a negative charge at phosphorus is not an absolute substrate requirement. The activities on the phosphatidic acid dimethyl ester of native enzyme and the Lys-69 mutant are lower than those on the corresponding lecithin, in contrast to the Phe-69 mutant, which has equal activities on both substrates. Our data suggest that in porcine pancreatic phospholipase A2 fixation of the phosphate group is achieved both by an interaction with the phenolic OH of Tyr-69 and by an interaction with the calcium ion. In the mutant Y69K the ε-NH2 group can play a role similar to that of the Tyr OH group in native PLA2. The smaller side chain of the Y69F mutant can interact with more bulky head groups, allowing for relatively high enzymatic activities on modified phospholipids. On the basis of these results, a reevaluation of the minimal substrate requirements of phospholipase A2 is presented.",
author = "Kuipers, {Oscar P.} and Nicolaas Dekker and Verheij, {Hubertus M.} and Haas, {Gerard H. de}",
note = "Relation: https://www.rug.nl/gbb/ date_submitted:2007 Rights: University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute",
year = "1990",
doi = "10.1021/bi00477a029",
language = "English",
volume = "29",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "AMER CHEMICAL SOC",
number = "25",

}

RIS

TY - JOUR

T1 - Activities of Native and Tyrosine-69 Mutant Phospholipases A2 on Phospholipid Analogues. A Reevaluation of the Minimal Substrate Requirements

AU - Kuipers, Oscar P.

AU - Dekker, Nicolaas

AU - Verheij, Hubertus M.

AU - Haas, Gerard H. de

N1 - Relation: https://www.rug.nl/gbb/ date_submitted:2007 Rights: University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute

PY - 1990

Y1 - 1990

N2 - The role of Tyr-69 of porcine pancreatic phospholipase A2 in substrate binding was studied with the help of proteins modified by site-directed mutagenesis and phospholipid analogues with a changed head-group geometry. Two mutants were used containing Phe and Lys, respectively, at position 69. Modifications in the phospholipids included introduction of a sulfur at the phosphorus (thionophospholipids), removal of the negative charge at phosphorus (phosphatidic acid dimethyl ester), and reduction (phosphonolipids) or extension (diacylbutanetriol choline phosphate) of the distance between the phosphorus and the acyl ester bond. Replacement of Tyr-69 by Lys reduces enzymatic activity, but the mutant enzyme retains both the stereospecificity and positional specificity of native phospholipase A2. The Phe-69 mutant not only hydrolyzes the Rp isomer of thionophospholipids more efficiently than the wild-type enzyme, but the Sp thiono isomer is hydrolyzed too, although at a low (~4%) rate. Phosphonolipids are hydrolyzed by native phospholipase A2 about 7 times more slowly than natural phospholipids, with retention of positional specificity and a (partial) loss of stereospecificity. The dimethyl ester of phosphatidic acid is degraded efficiently in a calcium-dependent and positional-specific way by native phospholipase A2 and by the mutants, indicating that a negative charge at phosphorus is not an absolute substrate requirement. The activities on the phosphatidic acid dimethyl ester of native enzyme and the Lys-69 mutant are lower than those on the corresponding lecithin, in contrast to the Phe-69 mutant, which has equal activities on both substrates. Our data suggest that in porcine pancreatic phospholipase A2 fixation of the phosphate group is achieved both by an interaction with the phenolic OH of Tyr-69 and by an interaction with the calcium ion. In the mutant Y69K the ε-NH2 group can play a role similar to that of the Tyr OH group in native PLA2. The smaller side chain of the Y69F mutant can interact with more bulky head groups, allowing for relatively high enzymatic activities on modified phospholipids. On the basis of these results, a reevaluation of the minimal substrate requirements of phospholipase A2 is presented.

AB - The role of Tyr-69 of porcine pancreatic phospholipase A2 in substrate binding was studied with the help of proteins modified by site-directed mutagenesis and phospholipid analogues with a changed head-group geometry. Two mutants were used containing Phe and Lys, respectively, at position 69. Modifications in the phospholipids included introduction of a sulfur at the phosphorus (thionophospholipids), removal of the negative charge at phosphorus (phosphatidic acid dimethyl ester), and reduction (phosphonolipids) or extension (diacylbutanetriol choline phosphate) of the distance between the phosphorus and the acyl ester bond. Replacement of Tyr-69 by Lys reduces enzymatic activity, but the mutant enzyme retains both the stereospecificity and positional specificity of native phospholipase A2. The Phe-69 mutant not only hydrolyzes the Rp isomer of thionophospholipids more efficiently than the wild-type enzyme, but the Sp thiono isomer is hydrolyzed too, although at a low (~4%) rate. Phosphonolipids are hydrolyzed by native phospholipase A2 about 7 times more slowly than natural phospholipids, with retention of positional specificity and a (partial) loss of stereospecificity. The dimethyl ester of phosphatidic acid is degraded efficiently in a calcium-dependent and positional-specific way by native phospholipase A2 and by the mutants, indicating that a negative charge at phosphorus is not an absolute substrate requirement. The activities on the phosphatidic acid dimethyl ester of native enzyme and the Lys-69 mutant are lower than those on the corresponding lecithin, in contrast to the Phe-69 mutant, which has equal activities on both substrates. Our data suggest that in porcine pancreatic phospholipase A2 fixation of the phosphate group is achieved both by an interaction with the phenolic OH of Tyr-69 and by an interaction with the calcium ion. In the mutant Y69K the ε-NH2 group can play a role similar to that of the Tyr OH group in native PLA2. The smaller side chain of the Y69F mutant can interact with more bulky head groups, allowing for relatively high enzymatic activities on modified phospholipids. On the basis of these results, a reevaluation of the minimal substrate requirements of phospholipase A2 is presented.

U2 - 10.1021/bi00477a029

DO - 10.1021/bi00477a029

M3 - Article

VL - 29

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 25

ER -

ID: 14674089