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 language | English (US) |
|---|---|
| Pages (from-to) | 4114-4120 |
| Number of pages | 7 |
| Journal | Biochemistry |
| Volume | 10 |
| Issue number | 22 |
| State | Published - 1971 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
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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 journal › Article
}
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.
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M3 - Article
C2 - 5168614
AN - SCOPUS:0015241519
VL - 10
SP - 4114
EP - 4120
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 22
ER -