Purification and mechanistic properties of an extracellular α-l-arabinofuranosidase from Monilinia fructigena

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Abstract
1. The .alpha.-L-arabinofuranosidase isoenzyme designated AFIII [Laborda, Archer, Fielding and Byrde (1974) J. Gen. Microbiol. 81, 151-163] was purified by sequential isoelectric focusing, hydrophobic chromatography, gel filtration and chromatofocusing. 2. The enzyme is a monomer of Mr 40000. 3. On inactivation of the enzyme with 3H-labelled 1-.alpha.-L-arabinofuranosylmethyl-3-p-nitrophenyltriazene, 0.64 mol of .alpha.-L-arabinofuranosylmethyl residues/mol of enzyme is estimated to become attached to protein. 5. Neither first-order nor second-order rate constants for hydrolyses of aryl .alpha.-L-arabinofuranosides are dependent upon leaving-group acidity [.beta.1g (V) = -0.16 .+-. 0.11; .beta.1g (V/K)= -0.11 .+-. 0.07; n = 7; .DELTA.pKa = 4.5] 6. Bond-breaking is nonetheless rate-limiting, as is shown by a value of 18(V) of 1.030 .+-. 0.007 for the hydrolysis of p-nitrophenyl arabinoside. 7. Proton-donation to the leaving group is thus far advanced at the rate-limiting transition state for this enzyme. 8. Four .alpha.-L-arabinofuranosyl pyridinium salts are substrates, and an approximate .beta.1g (V) value of -0.9 can be estimated. 9. The absolute rate enhancement with the 4-bromoisoquinolinium salt, 2.5 .times. 109, is comparable with that observed with pyranosidases. 10. Ring-opening mechanisms can therefore be dismissed, even though they are known in the acid-catalysed hydrolysis of arabinofuranosides.

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