Purification and mechanistic properties of an extracellular alpha-L-arabinofuranosidase from Monilinia fructigena.
1. The alpha-L-arabinofuranosidase isoenzyme designated AFIII [Laborda, Archer, Fielding & 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 40,000. 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 lg(V) = -0.16 +/- 0.11; Beta lg(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 lg(V) value of -0.9 can be estimated. 9. The absolute rate enhancement with the 4-bromoisoquinolinium salt, 2.5 X 10(9), 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.
Kelly
M A
MA
Department of Organic Chemistry, University of Bristol, U.K.
Sinnott
M L
ML
Herrchen
M
M
D001089:Arabinose / Q000031:analogs & derivatives / Q000378:metabolism; D001203:Ascomycota / Q000201:enzymology*; D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D005110:Extracellular Space / Q000201:enzymology; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D011726:Pyridinium Compounds / Q000378:metabolism; D013379:Substrate Specificity
D007527:Isoenzymes; D011726:Pyridinium Compounds; D001089:Arabinose; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase