Evaluation of the 'side door' in carboxylesterase-mediated catalysis and inhibition.
Abstract Structures of mammalian carboxylesterases (CEs) reveal the presence of a 'side door' that is proposed to act as an alternative pore for the trafficking of substrates and products. p-Nitrobenzyl esterase (pnb CE) from Bacillus subtilis exhibits close structural homology and a similar side-door domain as mammalian CEs. We investigated the role of a specific 'gate' residue at the side door (i.e., Leu 362) during pnb CE-catalyzed hydrolysis of model esters, pesticides, and lipids. Recombinant pnb CE proteins containing mutations at position 362 demonstrated markedly lower kcat and kcat/Km values. The mutation with the most significant impact on catalysis was the L362R mutant (kcat/Km was 22-fold lower). Moreover, the ability of the L362R mutant to be inhibited by organophosphates (OP) was also lower. Investigation into the altered catalytic proficiency using pH-activity studies indicated that the catalytic triad of the mutant enzyme was preserved. Furthermore, viscosity variation and carbamate inhibition experiments indicated that rates of substrate association and acylation/deacylation were lower. Finally, recombinant CEs were found to possess lipolytic activity toward cholesteryl oleate and 2-arachidonylglycerol. In summary, the L362R mutant CE markedly slowed the rate of ester hydrolysis and was less sensitive to OP inhibition. The apparent causes of the diminished catalysis are discussed.
Streit
Timothy M
TM
Center for Environmental Health Sciences, Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762, USA.
Borazjani
Abdolsamad
A
Lentz
Shellaine E
SE
Wierdl
Monika
M
Potter
Philip M
PM
Gwaltney
Steven R
SR
Ross
Matthew K
MK
D001412:Bacillus subtilis / Q000201:enzymology*; D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D004952:Esters / Q000378:metabolism; D007700:Kinetics; D050356:Lipid Metabolism; D020125:Mutation, Missense; D010575:Pesticides / Q000378:metabolism; D017434:Protein Structure, Tertiary
D004952:Esters; D010575:Pesticides; C091617:4-nitrobenzyl esterase; D002265:Carboxylic Ester Hydrolases