pH-dependence of the acetylcholinesterase - catalyzed oxygen exchange between acetate and water/purification and characterization of the low-molecular-weight acid phosphatase from bovine liver: Clarification of the roles of cysteine and histidine at the active site

Brian Donald Dayton, Purdue University

Abstract

Acetylcholinesterase (AChE) (acetylcholine hydrolase, E.C. 3.1.1.7) catalyzes ester hydrolysis through nucleophilic attack of an active-site serine on the acyl carbon of the substrate, acetylcholine, leading to the formation of an acylenzyme intermediate, with hydrolysis resulting in the production of choline and acetic acid. The first half of the reverse reaction--AChE-catalyzed attack on acetate--was studied over a wide range in pH. Homogeneous enzyme was obtained after chromatography using the synthetic affinity resin 9-(5-carboxypentylamino)acridine. The pH-dependence of AChE-catalyzed acetylthiocholine hydrolysis and its inhibition by acetate (1 M ionic strength) implicated one, or possibly two, active site residues in deacylation with pK$\sb{\rm a}$ values of 6.7 and 5.0. The pH-dependence of the enzyme-catalyzed exchange of oxygen between sodium (1-$\sp{13}$C,$\sp{18}$O$\sb2$) acetate and water suggested acetic acid as the preferred substrate for exchange, while rate data supported the role of an induced-fit rate-limiting step involving a "virtual transition state" in catalysis. The acid phosphatases (orthophosphoric monoester phosphohydrolase, E.C. 3.1.3.2) are a group of enzymes capable of hydrolyzing monobasic phosphate esters under acidic conditions (pH 4-7). Homogeneous low-molecular-weight acid phosphatase (BLAP) was purified 3500-fold from bovine liver tissue. Endogeneous proteolysis produced a major 14 kilodalton (kDa) fragment possessing phosphatase activity and a minor, inactive 12 kDa fragment eliminated as described. Physicochemical characterization of BLAP revealed a monomeric 17kDa protein with an isoelectric point (pI) of 6.3. Aggregation and inactivation of BLAP occurred at high (salt) and (protein), which was partially (30%) reactivated using 50 mM dithiothreitol + 4 M guanidinium$\cdot$HCl. O-Phospho-L-tyrosine phosphatase and phosphotransferase activities were reported; however, no reproducible BLAP-catalyzed exchange between phosphate(oxygen) and water was observed. The pH-dependence of p-nitrophenyl phosphate hydrolysis and its inhibition by phosphate suggested the importance of three residues in catalysis with pK$\sb{\rm a}$'s of 4.0-4.2, 6.2-6.3 and 7.4-7.6. Possible mechanistic roles for histidine, lysine and aspartate(glutamate) were suggested from chemical modification studies, as well as a functional role for at least one of three free sulfhydryl residues. Direct titration of the histidine C-2 proton, observed using $\sp1$H-NMR, defined its pK$\sb{\rm a}$ as 8.3. A model for the active site structure and mechanism was proposed.

Degree

Ph.D.

Advisors

Etten, Purdue University.

Subject Area

Biochemistry

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