The interplay of processivity, substrate inhibition and a secondary substrate binding site of an insect exo-beta-1,3-glucanase.
Abracris flavolineata midgut contains a processive exo-beta-glucanase (ALAM) with lytic activity against Saccharomyces cerevisiae, which was purified (yield, 18%; enrichment, 37 fold; specific activity, 1.89 U/mg). ALAM hydrolyses fungal cells or callose from the diet. ALAM (45 kDa; pI 5.5; pH optimum 6) major products with 0.6 mM laminarin as substrate are beta-glucose (61%) and laminaribiose (39%). Kinetic data obtained with laminaridextrins and methylumbelliferyl glucoside suggest that ALAM has an active site with at least six subsites. The best fitting of kinetic data to theoretical curves is obtained using a model where one laminarin molecule binds first to a high-affinity accessory site, causing active site exposure, followed by the transference of the substrate to the active site. The two-binding-site model is supported by results from chemical modifications of amino acid residues and by ALAM action in MUbetaGlu plus laminarin. Low laminarin concentrations increase the modification of His, Tyr and Asp or Glu residues and MUbetaGlu hydrolysis, whereas high concentrations abolish modification and inhibit MUbetaGlu hydrolysis. Our data indicate that processivity results from consecutive transferences of substrate between accessory and active site and that substrate inhibition arises when both sites are occupied by substrate molecules abolishing processivity.
Genta
Fernando A
FA
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, C.P. 26077, 05513-970, São Paulo, Brazil.
Dumont
Alexandra F
AF
Marana
Sandro R
SR
Terra
Walter R
WR
Ferreira
Clélia
C
D000818:Animals; D001665:Binding Sites; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology; D044643:Glucan 1,4-beta-Glucosidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005936:Glucans; D005960:Glucosides / Q000378:metabolism; D006110:Grasshoppers / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008956:Models, Chemical; D011134:Polysaccharides / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects
D005936:Glucans; D005960:Glucosides; D006902:Hydroxymercuribenzoates; D011134:Polysaccharides; C030601:4-hydroxymercuribenzoate; C014110:4-methylumbelliferyl glucoside; D006923:Hymecromone; C008247:laminaran; D044643:Glucan 1,4-beta-Glucosidase; D005022:Ethyldimethylaminopropyl Carbodiimide