Amino acids defining the acyl pocket of an invertebrate cholinesterase.
Amphioxus (Branchiostoma floridae) cholinesterase 2 (ChE2) hydrolyzes acetylthiocholine (AsCh) almost exclusively. We constructed a homology model of ChE2 on the basis of Torpedo californica acetylcholinesterase (AChE) and found that the acyl pocket of the enzyme resembles that of Drosophila melanogaster AChE, which is proposed to be comprised of Phe330 (Phe290 in T. californica AChE) and Phe440 (Val400), rather than Leu328 (Phe288) and Phe330 (Phe290), as in vertebrate AChE. In ChE2, the homologous amino acids are Phe312 (Phe290) and Phe422 (Val400). To determine if these amino acids define the acyl pocket of ChE2 and its substrate specificity, and to obtain information about the hydrophobic subsite, partially comprised of Tyr352 (Phe330) and Phe353 (Phe331), we performed site-directed mutagenesis and in vitro expression. The aliphatic substitution mutant F312I ChE2 hydrolyzes AsCh preferentially but also butyrylthiocholine (BsCh), and the change in substrate specificity is due primarily to an increase in k(cat) for BsCh; K(m) and K(ss) are also altered. F422L and F422V produce enzymes that hydrolyze BsCh and AsCh equally due to an increase in k(cat) for BsCh and a decrease in k(cat) for AsCh. Our data suggest that Phe312 and Phe422 define the acyl pocket. We also screened mutants for changes in sensitivity to various inhibitors. Y352A increases the sensitivity of ChE2 to the bulky inhibitor ethopropazine. Y352A decreases inhibition by BW284c51, consistent with its role as part of the choline-binding site. Aliphatic replacement mutations produce enzymes that are more sensitive to inhibition by iso-OMPA, presumably by increasing access to the active site serine. Y352A, F353A and F353V make ChE2 considerably more resistant to inhibition by eserine and neostigmine, suggesting that binding of these aromatic inhibitors is mediated by pi-pi or cation-pi interactions at the hydrophobic site. Our results also provide information about the aromatic trapping of the active site histidine and the inactivation of ChE2 by sulfhydryl reagents.
Pezzementi
Leo
L
Division of Science and Mathematics, Birmingham-Southern College, Box 549022, Birmingham, AL 35254, USA. lpezzeme@bsc.edu
Johnson
Kimberly
K
Tsigelny
Igor
I
Cotney
Justin
J
Manning
Elizabeth
E
Barker
Andrew
A
Merritt
Sarah
S
D000122:Acetylthiocholine / Q000378:metabolism; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002092:Butyrylthiocholine / Q000378:metabolism; D019556:COS Cells; D002800:Cholinesterase Inhibitors / Q000378:metabolism; D002802:Cholinesterases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D020128:Inhibitory Concentration 50; D007448:Invertebrates / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013696:Temperature
D000596:Amino Acids; D002800:Cholinesterase Inhibitors; D013438:Sulfhydryl Compounds; D000122:Acetylthiocholine; D002092:Butyrylthiocholine; C116697:cholinesterase 2; D002802:Cholinesterases