| Glycosyl hydrolases, capable of selectively hydrolyzing glycosidic bonds, possess enormous potential to participate in the production of fuels and the treatment of a wide range of illnesses. The exo-glucanases are a unique class of the glycosyl hydrolases known as cellobiohydrolases (CBHI and CBHII) because of their ability to hydrolyze cellulose into cellobiose units. The possibility that CBHI and CBHII utilize the free energy of the hydrolysis of a glycosidic link to disrupt the cellulose crystallite, and should therefore be able to catalyze the reverse reaction to drive the thermodynamically disfavored synthesis of a {dollar}beta{dollar}-glucan link using cellulose crystallization energy, was investigated. It has been demonstrated that incubation of {dollar}sp3{dollar}H-cellobiose with Acetobacter xylinum cellulose and CBHII results in the incorporation of radioactive cellobiose into the cellulose in a time and concentration dependent manner, whereas incubation in the absence of the enzyme or in the presence of BSA, results in a far lower rate of incorporation. CBHI, on the other hand, demonstrated a short pulse of incorporation followed by progressive loss of radioactivity which may be attributed to "snap-back", in which a molecule of radioactive cellobiose can transfer itself to the glycosyl enzyme intermediate. Additionally, {dollar}DeltaDelta{dollar}G{dollar}sp{lcub}ddagger{rcub}{dollar} values estimating the extent of oxocarbonium ion character in the transition state of CBHI were found to range from 32.15 kJ/mole to 34.88 kJ/mole. CBHI, also, demonstrated that, in addition to its primary role in the hydrolysis of cellulose, it participates in transglycosylation which implies that deglycosylation is the rate limiting step. Finally, calculated inactivation parameters were combined with a molecular modeling simulation in order to study the effects of the Withers inactivator, 2,4-DNP-2-deoxy-2-fluoro-{dollar}alpha{dollar}-cellobioside on the mechanism of CBHII. The ability of CBHII to adopt a double displacement mechanism instead of the traditional single displacement mechanism was investigated. |
