A detailed mechanistic investigation of the exoglycanase from Cellulomonas fimi
Creators: name: Tull, Dedreia L.
Publisher: name: University of British Columbia
Year: 2009

description: The exoglycanase from Cellulomonas fimi catalyses the hydrolysis of cello oligosaccharides to cellobiose as well as the hydrolysis of xylan and aryl β-glycosides (Gilkes et al (1984) J. Biol. Chem. 259, 10455). Its mechanism of action is thought to involve a double displacement reaction which is investigated here through detailed kinetic studies of the native enzyme and point mutants with a range of aryl β-glycosides, and through inactivation studies with 2-deoxy- and 2-deoxyfluoro-glycoside mechanism-based inactivators and the affinity label, N-bromoacetyl cellobiosylamine. A pH study of the native enzyme revealed ionisations of PKa = 4.1 and 7.7 in the free enzyme, likely corresponding to the catalytic nucleophile and the acid-base catalyst, respectively. The large secondary deuterium kinetic isotope effects measured on both steps for the glucosides and on the deglycosylation step for the cellobiosides reveal significant oxocarbonium ion character at the corresponding transition states, thus suggesting substantial C-O bond cleavage and little nucleophilic preassociation. By contrast, the relatively small secondary deuterium kinetic isotope effect and the small Broensted constant measured on the glycosylation step for the cellobiosides suggest that the cellobiosylation transition state is less highly charged than the glucosylation transition state. These studies suggest that the primary function of the distal glucosyl moiety of the cellobiosides is to increase the rate of glycosylation, likely through improved acid catalysis and greater nucleophile preassociation, without affecting its rate of deglycosylation. The greater rates of hydrolysis of the xylo-sugars, relative to those for the gluco-sugars, indicate that the substrate preference of C. fimi exoglycanase increases in the order glucosides &lt;xylosides &lt;cellobiosides &lt;xylobiosides and that the C-5 hydroxymethyl group is slightly inhibitory to catalysis. The role of the C-2 hydroxyl group was probed using 2,4-dinitrophenyl 2-deoxy-2-fluoro cellobioside (2F-DNPC) and cellobial (a 2-deoxycellobiose analogue). Rates of hydrolysis of the 2-deoxyfluorocellobiosyl- and 2-deoxycellobiosyl-enzymes are 10⁷ and10⁶-fold lower respectively, than that for the cellobiosyl-enzyme, indicating that the C-2 hydroxyl group is necessary for catalysis and that it contributes a minimum of -9 kcal/mole of stabilisation energy to the transition state. Electrospray ionisation mass spectrometry (ESI-MS) of the 2F-DNPC-inactivated enzyme provided evidence for the covalent nature of the glycosyl-enzyme intermediate while ¹⁹FNMR analysis of this 2FCb-enzyme and the 2-deoxy-2-fluoro 4-O-(f-glucosyl)- β-mannosyl fluoride (2F-GMF) -inactivated enzyme provided evidence for the α-anomeric stereochemistry of the intermediate. The catalytic nucleophile involved in C. fimi exoglycanase-catalysed hydrolysis of the cellobiosides was identified as Glu 233 by use of tandem MS techniques and 2F-DNPC and cellobial. Kinetic analysis of the Glu233Asp mutant revealed that pulling the catalytic nucleophile 1 Å away from the reacting anomeric centre reduces the rates of glycosylation and deglycosylation —4 x10³-fold. ESI-MS analysis of N-bromoacetyl cellobiosylamine-inactivated C. fimi exoglycanase reveals that one mole of N-acetyl cellobiosylamine is incorporated per mole of enzyme. The labeled residue was identified as Glu 127 by use of a combination of MS techniques. This residue has recently been suggested to be the acid-base catalyst based on kinetic analysis of mutants (MacLeod et al (1994) Biochemistry 33, 6571). More detailed kinetic analysis of the Glul27Ala mutant revealed rate reductions of 200-300 fold on the deglycosylation step while the rate reductions on the glycosylation step are dependent on the leaving group ability of the phenolate. The larger Broensted constant seen with the Glul27Ala mutant compared to that for the native enzyme reflects greater negative charge accumulation on the leaving phenolate at the glycosylation transition state for the Glul27Ala mutant. These results are consistent with the role of Glu 127 as the acid-base catalyst. These structural findings are completely consistent with the recently solved X-ray crystal structure of the catalytic domain of C.fimi exoglycanase (White et al (1994) Biochemistry 33, 12546).