Studies of catalysis by acetylcholinesterase. I. Fluorescent titration with a carbamoylating agent

Terrone L. Rosenberry, Sidney A. Bernhard

Research output: Contribution to journalArticle

82 Citations (Scopus)

Abstract

The reaction of acetylcholinesterase with the N-methyl-7-hydroxyquinolinium iodide ester of dimethylcarbamic acid has been followed spectrofluorometrically. In an excess of substrate the observed rate of carbamoylation of the enzyme is strictly first order and is much greater than the concentration-independent rate of decarbamoylation, allowing a titration of enzyme active sites. The method is sensitive to enzyme normalities in the nanomolar region, and from the turnover number and the enzyme normality, the number of active sites per molecule can be calculated. The effects of inhibitors on both the carbamoylation and decarbamoylation rates have been analyzed. Competitive inhibition constants for the inhibition of carbamoylation are the same as those obtained for the inhibition of enzyme-catalyzed acetic acid ester hydrolysis and support the idea that the same enzyme active site is involved in both cases. Some competitive inhibitors of acetylcholine hydrolysis and of carbamoylation are bound to the carbamoyl-enzyme as well as to the free native enzyme. These "inhibitors" are positive effectors of the rate of carbamoyl-enzyme hydrolysis and thereby support previous proposals of a second allosteric binding site.

Original languageEnglish (US)
Pages (from-to)4114-4120
Number of pages7
JournalBiochemistry
Volume10
Issue number22
StatePublished - 1971
Externally publishedYes

Fingerprint

Acetylcholinesterase
Catalysis
Titration
Enzymes
Hydrolysis
Catalytic Domain
Allosteric Site
Iodides
Acetylcholine
Esters
Acetates
Binding Sites
Molecules
Acids
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Studies of catalysis by acetylcholinesterase. I. Fluorescent titration with a carbamoylating agent. / Rosenberry, Terrone L.; Bernhard, Sidney A.

In: Biochemistry, Vol. 10, No. 22, 1971, p. 4114-4120.

Research output: Contribution to journalArticle

Rosenberry, Terrone L. ; Bernhard, Sidney A. / Studies of catalysis by acetylcholinesterase. I. Fluorescent titration with a carbamoylating agent. In: Biochemistry. 1971 ; Vol. 10, No. 22. pp. 4114-4120.
@article{4cde999e8694447bac7d3998c3c1eee5,
title = "Studies of catalysis by acetylcholinesterase. I. Fluorescent titration with a carbamoylating agent",
abstract = "The reaction of acetylcholinesterase with the N-methyl-7-hydroxyquinolinium iodide ester of dimethylcarbamic acid has been followed spectrofluorometrically. In an excess of substrate the observed rate of carbamoylation of the enzyme is strictly first order and is much greater than the concentration-independent rate of decarbamoylation, allowing a titration of enzyme active sites. The method is sensitive to enzyme normalities in the nanomolar region, and from the turnover number and the enzyme normality, the number of active sites per molecule can be calculated. The effects of inhibitors on both the carbamoylation and decarbamoylation rates have been analyzed. Competitive inhibition constants for the inhibition of carbamoylation are the same as those obtained for the inhibition of enzyme-catalyzed acetic acid ester hydrolysis and support the idea that the same enzyme active site is involved in both cases. Some competitive inhibitors of acetylcholine hydrolysis and of carbamoylation are bound to the carbamoyl-enzyme as well as to the free native enzyme. These {"}inhibitors{"} are positive effectors of the rate of carbamoyl-enzyme hydrolysis and thereby support previous proposals of a second allosteric binding site.",
author = "Rosenberry, {Terrone L.} and Bernhard, {Sidney A.}",
year = "1971",
language = "English (US)",
volume = "10",
pages = "4114--4120",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "22",

}

TY - JOUR

T1 - Studies of catalysis by acetylcholinesterase. I. Fluorescent titration with a carbamoylating agent

AU - Rosenberry, Terrone L.

AU - Bernhard, Sidney A.

PY - 1971

Y1 - 1971

N2 - The reaction of acetylcholinesterase with the N-methyl-7-hydroxyquinolinium iodide ester of dimethylcarbamic acid has been followed spectrofluorometrically. In an excess of substrate the observed rate of carbamoylation of the enzyme is strictly first order and is much greater than the concentration-independent rate of decarbamoylation, allowing a titration of enzyme active sites. The method is sensitive to enzyme normalities in the nanomolar region, and from the turnover number and the enzyme normality, the number of active sites per molecule can be calculated. The effects of inhibitors on both the carbamoylation and decarbamoylation rates have been analyzed. Competitive inhibition constants for the inhibition of carbamoylation are the same as those obtained for the inhibition of enzyme-catalyzed acetic acid ester hydrolysis and support the idea that the same enzyme active site is involved in both cases. Some competitive inhibitors of acetylcholine hydrolysis and of carbamoylation are bound to the carbamoyl-enzyme as well as to the free native enzyme. These "inhibitors" are positive effectors of the rate of carbamoyl-enzyme hydrolysis and thereby support previous proposals of a second allosteric binding site.

AB - The reaction of acetylcholinesterase with the N-methyl-7-hydroxyquinolinium iodide ester of dimethylcarbamic acid has been followed spectrofluorometrically. In an excess of substrate the observed rate of carbamoylation of the enzyme is strictly first order and is much greater than the concentration-independent rate of decarbamoylation, allowing a titration of enzyme active sites. The method is sensitive to enzyme normalities in the nanomolar region, and from the turnover number and the enzyme normality, the number of active sites per molecule can be calculated. The effects of inhibitors on both the carbamoylation and decarbamoylation rates have been analyzed. Competitive inhibition constants for the inhibition of carbamoylation are the same as those obtained for the inhibition of enzyme-catalyzed acetic acid ester hydrolysis and support the idea that the same enzyme active site is involved in both cases. Some competitive inhibitors of acetylcholine hydrolysis and of carbamoylation are bound to the carbamoyl-enzyme as well as to the free native enzyme. These "inhibitors" are positive effectors of the rate of carbamoyl-enzyme hydrolysis and thereby support previous proposals of a second allosteric binding site.

UR - http://www.scopus.com/inward/record.url?scp=0015241519&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0015241519&partnerID=8YFLogxK

M3 - Article

VL - 10

SP - 4114

EP - 4120

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 22

ER -