SOME DETAILS OF THE REACTION-MECHANISM OF GLUCOAMYLASE FROM ASPERGILLUS-NIGER - KINETIC AND STRUCTURAL STUDIES ON TRP52-]PHE AND TRP317-]PHE MUTANTS

Presteady and steady-state kinetic results on the interactions of a wi ld-type, and the mutant glucoamylases Trp52-->Phe and Trp317-->Phe, fr om Aspergillus niger with maltose, maltotriose and maltotetraose have been obtained and analyzed. The results are compared with previous one s on the mutants, Trp120-->Phe and Glu180-->Gln, and with results obta ined from structure energy minimization calculations based on known th ree-dimensional structural data. All results are in accordance with a three-step reaction model involving two steps in the substrate binding and a rate-determining catalytic step. Trp317 and Glu180 belong to di fferent subsites. but are placed on the same flank of the active site (beta-flank). The Trp317-->Phe and the Glu180-->Gln mutants show almos t identical kinetic results: weakening of the substrate binding, mainl y caused by changes in the second reaction step, and practically no ch ange of the catalytic rate. Structure energy minimization calculations show that the same loss of Arg305 and Glu180 hydrogen bonds to the su bstrate occurs in the Michaelis complexes of each of these mutants. Th ese results indicate that important interactions of the active site ma y be better understood from a consideration of its flanks rather than of its subsites. The results further indicate differences in the subst rate binding mode of maltose and of longer substrates. Trp52 and Trp12 0 each interact with the catalytic acid, Glu179, and are placed on the flank (alpha-flank) of the active site opposite to Trp317, Arg305 and Glu180. Also the Trp52-->Phe and Trp120-->Phe mutants show kinetic re sults similar to each other. The catalytic rates are strongly reduced and the substrates are bound more strongly, mainly as a result of the formation of a more stable complex in the second reaction step. All to gether, the substrate binding mechanism seems to involve an initial en zyme-substrate complex, in which the beta-flank plays a minor role, ex cept for maltose binding; this is followed by a conformational change, in which hydrogen bends to Arg305 and Glu180 of the beta-flank are es tablished and the correct alignment on the alpha-flank of Glu179. the general acid catalyst, governed by its flexible interactions with Trp5 2 and Trp120, occurs.