The kinetics of p-nitrophenyl-β-D-cellobioside hydrolysis and transglycosylation by Thermobifida fusca Cel5Acd.
The hydrolysis of p-nitrophenyl-β-1,4-cellobioside (pNP-G2) by the catalytic domain of the retaining-family 5-2 endocellulase Cel5A from Thermobifida fusca (Cel5Acd) was studied. The dominant reaction pathway involves hydrolysis of the aglyconic bond, producing cellobiose (G2) and a 'reporter' species p-nitrophenol (pNP), which was monitored spectrophotometrically to track the reaction. We also detected the production of cellotriose (G3) and p-nitrophenyl-glucoside (pNP-G1), confirming the presence of a competing transglycosylation pathway. We use a mechanistic model of hydrolysis and transglycosylation to derive an expression for the rate of pNP-formation as a function of enzyme concentration, substrate concentration, and several lumped kinetics parameters. The derivation assumes that the quasi-steady-state assumption (QSSA) applies for three intermediate species in the mechanism; we determine conditions under which this assumption is rigorously justified. We integrate the rate expression and compare its integral form to pNP-versus-time data collected for a range of enzyme and substrate concentrations. The integral comparison gives a stringent test of the mechanistic model, and it serves to quantify the lumped kinetics parameters with good statistical precision, particularly a previously unidentified parameter that determines the selectivity of hydrolysis versus transglycosylation. The integrated rate expression accounts well for pNP-versus-time data under all circumstances we have investigated.
Dingee
John W
JW
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14850, United States.
Anton
A Brad
AB
D000192:Actinomycetales / Q000201:enzymology*; D020134:Catalytic Domain; D002475:Cellobiose / Q000494:pharmacology; D002480:Cellulase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D005960:Glucosides / Q000378:metabolism*; D006031:Glycosylation; D006868:Hydrolysis; D007700:Kinetics; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D005960:Glucosides; D002475:Cellobiose; C034170:4-nitrophenyl beta-cellobioside; C407024:endoglucanase Cel5A; D002480:Cellulase; D005947:Glucose