A substrate-induced switch in the reaction mechanism of a thermophilic esterase: kinetic evidences and structural basis.
The reaction mechanism of the esterase 2 (EST2) from Alicyclobacillus acidocaldarius was studied at the kinetic and structural level to shed light on the mechanism of activity and substrate specificity increase previously observed in its double mutant M211S/R215L. In particular, the values of kinetic constants (k1, k(-1), k2, and k3) along with activation energies (E1, E(-1), E2, and E3) were measured for wild type and mutant enzyme. The previously suggested substrate-induced switch in the reaction mechanism from kcat=k3 with a short acyl chain substrate (p-nitrophenyl hexanoate) to kcat=k2 with a long acyl chain substrate (p-nitrophenyl dodecanoate) was validated. The inhibition afforded by an irreversible inhibitor (1-hexadecanesulfonyl chloride), structurally related to p-nitrophenyl dodecanoate, was studied by kinetic analysis. Moreover the three-dimensional structure of the double mutant bound to this inhibitor was determined, providing essential information on the enzyme mechanism. In fact, structural analysis explained the observed substrate-induced switch because of an inversion in the binding mode of the long acyl chain derivatives with respect to the acyl- and alcohol-binding sites.
De Simone
Giuseppina
G
Istituto di Biostrutture e Bioimmagini-Consiglio Nazionale delle Ricerche, via Mezzocannone 6, 80134 Naples, Italy.
Mandrich
Luigi
L
Menchise
Valeria
V
Giordano
Valeria
V
Febbraio
Ferdinando
F
Rossi
Mosè
M
Pedone
Carlo
C
Manco
Giuseppe
G
D000438:Alcohols / Q000737:chemistry; D001419:Bacteria / Q000201:enzymology*; D001665:Binding Sites; D002208:Caproates / Q000737:chemistry; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry*; D002384:Catalysis; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D007848:Laurates / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D009578:Nitrobenzenes / Q000737:chemistry; D011485:Protein Binding; D013379:Substrate Specificity; D013450:Sulfones / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors
C484324:1-hexadecanesulfonyl chloride; C484326:4-nitrophenyl dodecanoate; C484325:4-nitrophenyl hexanoate; D000438:Alcohols; D002208:Caproates; D007848:Laurates; D009578:Nitrobenzenes; D013450:Sulfones; D002265:Carboxylic Ester Hydrolases