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2.
Biochemistry ; 40(18): 5422-32, 2001 May 08.
Article in English | MEDLINE | ID: mdl-11331006

ABSTRACT

Phosphatidylinositol-specific phospholipase C (PI-PLC) catalyzes the cleavage of the P-O bond in phosphatidylinositol via intramolecular nucleophilic attack of the 2-hydroxyl group of inositol on the phosphorus atom. Our earlier stereochemical and site-directed mutagenesis studies indicated that this reaction proceeds by a mechanism similar to that of RNase A, and that the catalytic site of PI-PLC consists of three major components analogous to those observed in RNase A, the His32 general base, the His82 general acid, and Arg69 acting as a phosphate-activating residue. In addition, His32 is associated with Asp274 in forming a catalytic triad with inositol 2-hydroxyl, and His82 is associated with Asp33 in forming a catalytic diad. The focus of this work is to provide a global view of the mechanism, assess cooperation between various catalytic residues, and determine the origin of enzyme activation by the hydrophobic leaving group. To this end, we have investigated kinetic properties of Arg69, Asp33, and His82 mutants with phosphorothioate substrate analogues which feature leaving groups of varying hydrophobicity and pK(a). Our results indicate that interaction of the nonbridging pro-S oxygen atom of the phosphate group with Arg69 is strongly affected by Asp33, and to a smaller extent by His82. This result in conjunction with those obtained earlier can be rationalized in terms of a novel, dual-function triad comprised of Arg69, Asp33, and His82 residues. The function of this triad is to both activate the phosphate group toward the nucleophilic attack and to protonate the leaving group. In addition, Asp33 and His82 mutants displayed much smaller degrees of activation by the fatty acid-containing leaving group as compared to the wild-type (WT) enzyme, and the level of activation was significantly reduced for substrates featuring the leaving group with low pK(a) values. These results strongly suggest that the assembly of the above three residues into the fully catalytically competent triad is controlled by the hydrophobic interactions of the enzyme with the substrate leaving group.


Subject(s)
Arginine/chemistry , Asparagine/chemistry , Catalytic Domain , Histidine/chemistry , Organophosphates/chemistry , Type C Phospholipases/chemistry , Amino Acid Substitution/genetics , Arginine/genetics , Asparagine/genetics , Bacillus cereus/enzymology , Binding Sites/genetics , Catalysis , Catalytic Domain/genetics , Histidine/genetics , Hydrolysis , Inositol Phosphates/chemistry , Phosphatidylinositol Diacylglycerol-Lyase , Phosphoinositide Phospholipase C , Ribonuclease, Pancreatic/chemistry , Glycine max/enzymology , Structure-Activity Relationship , Substrate Specificity/genetics , Sulfur/chemistry , Thionucleotides/chemistry , Type C Phospholipases/genetics
3.
Biochemistry ; 40(18): 5433-9, 2001 May 08.
Article in English | MEDLINE | ID: mdl-11331007

ABSTRACT

Phosphatidylinositol-specific phospholipase C (PI-PLC) has been proposed previously to employ a catalytic mechanism highly reminiscent of that of ribonuclease A (RNase A). Both catalytic sites are comprised of two histidine side chains acting as a general base-general acid pair and a phosphate-activating residue: an arginine in the case of PI-PLC and a lysine in RNase A. Despite these structural similarities, the PI-PLC reaction is slowed 10(5)-fold upon substitution of one of the phosphate nonbridging oxygen atoms with sulfur, whereas a much smaller effect is observed in the analogous RNase A reaction. Here, we report a systematic study of this property in PI-PLC, conducted by means of site-directed chemical modification of a cysteine residue replacing the arginine at position 69. The results show that mutant enzymes featuring bidentate side chains at this position display significantly higher activity, higher thio effects, and greater stereoselectivity than do those with monodentate side chains. The results suggest that the bidentate nature of Arg69 is the origin of the large thio effects and stereoselectivity in PI-PLC. We propose that in addition to binding the phosphate, the function of arginine 69 is to bring the phosphate group and the 2-OH group of inositol into proximity and to induce proper alignment for nucleophilic attack, and possibly to lower the pK(a) of the 2-OH. The results presented here could be important to mechanisms of phosphoryl transfer enzymes in general, suggesting that a major part of thio effects observed in enzymatic phosphoryl transfer reactions can originate from factors other than direct interaction between a side chain and a phosphate group, and caution the use of the absolute magnitude of the thio effect as an indicator of the strength of such interactions.


Subject(s)
Bacterial Proteins/chemistry , Sulfhydryl Compounds/chemistry , Type C Phospholipases/chemistry , Amino Acid Substitution/genetics , Animals , Arginine/chemistry , Arginine/genetics , Bacterial Proteins/genetics , Cysteine/chemistry , Cysteine/genetics , Humans , Ligands , Models, Chemical , Mutagenesis, Site-Directed , Phosphatidic Acids/chemistry , Phosphatidylinositol Diacylglycerol-Lyase , Phosphatidylinositols/chemistry , Phosphoinositide Phospholipase C , Rats , Ribonuclease, Pancreatic/chemistry , Stereoisomerism , Structure-Activity Relationship , Substrate Specificity/genetics , Thionucleotides/chemistry , Type C Phospholipases/genetics
4.
J Orthop Sports Phys Ther ; 8(11): 537-41, 1987.
Article in English | MEDLINE | ID: mdl-18797024

ABSTRACT

Reports in the literature appear to support the effectiveness of electrical stimulation as a means of increasing strength in normal muscles. The objective of this study was to measure and compare quadriceps muscle torque, isometrically and isokinetically, in three groups: 1) control group subjects (N = 9) did not alter their daily activities for 5 weeks, 2) isometric exercise group subjects (N = 10) exercised their quadriceps femoris muscle using maximum voluntary isometric exercises 3 times per week for 5 weeks, and 3) electrical stimulation group subjects (N = 10) who had 10 quadriceps femoris muscle contractions produced by electrical stimulation, 3 times per week for 5 weeks. No strength changes were noted in the control group; ANOVA revealed significant strength increases (P < 0.05) for both the electrical stimulation and isometric exercise groups. J Orthop Sports Phys Ther 1987;8(11):537-541.

5.
J Med Educ ; 52(10): 859-60, 1977 Oct.
Article in English | MEDLINE | ID: mdl-143533
6.
Med Biol Illus ; 20(2): 123-4, 1970 Apr.
Article in English | MEDLINE | ID: mdl-4195251
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