Human Carboxylesterase 1 (hCES1) is the key liver microsomal enzyme responsible for detoxification and metabolism of a variety of clinical drugs. To analyse the role of the single N-linked glycan on the structure and activity of the enzyme, authentically glycosylated and aglycosylated hCES1, generated by mutating asparagine 79 to glutamine, were produced in human embryonic kidney cells. Purified enzymes were shown to be predominantly trimeric in solution by analytical ultracentrifugation. The purified aglycosylated enzyme was found to be more active than glycosylated hCES1 and analysis of enzyme kinetics revealed that both enzymes exhibit positive cooperativity. Crystal structures of hCES1 a catalytically inactive mutant (S221A) and the aglycosylated enzyme were determined in the absence of any ligand or substrate to high resolutions (1.86 A, 1.48 A and 2.01 A, respectively). Superposition of all three structures showed only minor conformational differences with a root mean square deviations of around 0.5 A over all Calpha positions. Comparison of the active sites of these un-liganded enzymes with the structures of hCES1-ligand complexes showed that side-chains of the catalytic triad were pre-disposed for substrate binding. Overall the results indicate that preventing N-glycosylation of hCES1 does not significantly affect the structure or activity of the enzyme.
Esterases and deacetylases active on carbohydrate ligands have been classified into 14 families based upon amino acid sequence similarities. Enzymes from carbohydrate esterase family seven (CE-7) are unusual in that they display activity towards both acetylated xylooligosaccharides and the antibiotic, cephalosporin C. The 1.9A structure of the multifunctional CE-7 esterase (hereinafter CAH) from Bacillus subtilis 168 reveals a classical alpha/beta hydrolase fold encased within a 32 hexamer. This is the first example of a hexameric alpha/beta hydrolase and is further evidence of the versatility of this particular fold, which is used in a wide variety of biological contexts. A narrow entrance tunnel leads to the centre of the molecule, where the six active-centre catalytic triads point towards the tunnel interior and thus are sequestered away from cytoplasmic contents. By analogy to self-compartmentalising proteases, the tunnel entrance may function to hinder access of large substrates to the poly-specific active centre. This would explain the observation that the enzyme is active on a variety of small, acetylated molecules. The structure of an active site mutant in complex with the reaction product, acetate, reveals details of the putative oxyanion binding site, and suggests that substrates bind predominantly through non-specific contacts with protein hydrophobic residues. Protein residues involved in catalysis are tethered by interactions with protein excursions from the canonical alpha/beta hydrolase fold. These excursions also mediate quaternary structure maintenance, so it would appear that catalytic competence is only achieved on protein multimerisation. We suggest that the acetyl xylan esterase (EC 3.1.1.72) and cephalosporin C deacetylase (EC 3.1.1.41) enzymes of the CE-7 family represent a single class of proteins with a multifunctional deacetylase activity against a range of small substrates.
Title: Diagnostic value of acetylcholinesterase/butyrylcholinesterase ratio in Hirschsprung's disease [letter] Bonham JR, Dale G, Scott DJ, Waggett J Ref: American Journal of Clinical Pathology, 90:520, 1988 : PubMed
Title: Acetylcholinesterase activity in rectal biopsies: an assessment of its diagnostic value in Hirschsprung's disease [letter] Bonham JR, Dale G, Scott DJ, Wagget J Ref: J Pediatr Gastroenterol Nutr, 7:298, 1988 : PubMed
Title: The characterisation of molecular forms of acetylcholinesterase in Hirschsprung's disease Bonham JR, Dale G, Scott DJ, Wagget J, Atack JR Ref: Clinica Chimica Acta, 171:263, 1988 : PubMed
Aganglionic rectal tissue from patients with Hirschsprung's disease contains four forms of acetylcholinesterase; the major component has a sedimentation coefficient in the region of 10.0S. Results from gel filtration confirm these findings and, when used in conjunction with the sedimentation data, allow the determination of the molecular mass of these forms. The four species of acetylcholinesterase include: monomer, G1, 74 kDa dimer, G2, 131 kDa; tetramer G4, 275 kDa and an asymmetric form, A12, 811 kDa. Evidence is provided which shows that the major form, G4 interacts with the detergent Triton X-100. Selective measurement of G4-AChE using a suitable assay may provide the basis for improving the existing means of diagnosis of Hirschsprung's disease.
Title: A 7-year study of the diagnostic value of rectal mucosal acetylcholinesterase measurement in Hirschsprung's disease Bonham JR, Dale G, Scott DJ, Wagget J Ref: J Pediatr Surg, 22:150, 1987 : PubMed
Over a 7-year period, 213 children were investigated for failure to pass meconium or for chronic constipation. Of these, 45 were confirmed to have Hirschsprung's disease; in this group the acetylcholinesterase activity in rectal biopsy tissue was significantly increased (P less than .001; mean 34.2, 95% confidence limits, 8.6 to 95.2) units g-1 when compared with the non-Hirschsprung's group (mean 6.6, 95% confidence limits, 2.0 to 15.9 units g-1). By expressing the acetylcholinesterase activity as a percentage of the total cholinesterase activity it is possible to compensate for evaporative weight loss and the combination of these two measurements improves the overall diagnostic value of the test. There were no false-positive and only two false-negative results.
Acetylcholinesterase (AChE) activity was measured in rectal biopsy specimens obtained from 68 children aged between 2 days and 14 1/2 years in whom Hirschsprung's disease was suspected. The diagnosis was subsequently established in 12; in these, the mean AChE activity was found to be 30.5 X 10(-7) units/g tissue (range 16.9 to 63.0). The 56 non-Hirschsprung cases had a mean of 5.0 X 10(-7) units/g tissue (S.D. 2.2), the highest value in this group being 10.9. The results were unaffected by age, sex, nature of biopsy procedure, or the presence of blood. It is suggested that the assay of AChE activity in rectal biopsy material is a simple and quick procedure that is useful in the diagnosis of Hirschsprung's disease.
