Comparison of atypical and usual human serum cholinesterase. Purification, number of active sites, substrate affinity, and turnover number.

O Lockridge,B.N La Du

doi:10.1016/s0021-9258(17)38214-5

Abstract

Atypical and usual human serum cholinesterases were purified and studied with the fluorescent probe, N-methyl-(7-dimethylcarbamoxy)quinolinium iodide. Four active sites per tetramer were found in each enzyme. The turnover numbers of usual and atypical cholinesterases were the same: 15,000 mumol of benzoylcholine hydrolyzed/min/mumol of active site; 48,000 min-1 for o-nitrophenylbutyrate; and 0.0025 min-1 for N-methyl-(7-dimethylcarbamoxy)quinolinium iodide. They had identical rate constants for carbamylation, (5.0 min-1) and for decarbamylation (0.15 h-1). The major difference between the two genetically determined forms of the enzyme was substrate affinity, KD being 0.16 mM for usual and 5.4 mM for atypical cholinesterase, for the fluorescent probe substrate. Km for the uncharged ester, o-nitrophenylbutyrate, was 0.14 mM for both enzymes, whereas Km for benzoylcholine was 0.005 mM for usual and 0.024 mM for atypical cholinesterase. We interpret these data to mean that the two enzymes differ only in the structure of their anionic site.

Highlights

IntroductionLA Du From the Pharmacology Department, M6322 Medical Medical School, Ann Arbor, Michigan 48109
In order to determine how atypical human serum cholinesterase differed from usual serum choline&erase, we purified the enzyme from four usual and two atypical donors and studied the purified enzymes with the fluorescent probe substrate
We found the rate constants for carbamylation and decarbamylation to be the same for both enzymes and, the turnover number for this compound, N-methyl-(7

Summary

Introduction

LA Du From the Pharmacology Department, M6322 Medical Medical School, Ann Arbor, Michigan 48109. Four active sites per tetramer were found in each enzyme. The turnover numbers of usual and atypical cholinesterases were the same: 15,000 pmol of benzoylcholine hydrol~zedlminl~mol of active site; 48,000 min-’ for o-nitrophenylbutyrate; and. The major difference between the two genetically determined forms of the enzyme was substrate affinity, Ku being. 0.16 mM for usual and 5.4 mM for atypical cholinesterase, for the fluorescent probe substrate. K,,, for the uncharged ester, o-nitrophenylbutyrate, was 0.14 mM for both enzymes, whereas K,, for benzoylcholine was 0.005 mM for usual and. We interpret these data to mean that the two enzymes differ only in the structure of their anionic site

Methods

Results

Conclusion