Torben P. Frandsen

Physicochemical characterisation of the two active site mutants Trp52→Phe and Asp55→Val of glucoamylase from Aspergillus niger

Glucoamylase 1 (GA1) from Aspergillus niger is a multidomain starch hydrolysing enzyme that consists of a catalytic domain and a starch-binding domain connected by an O-glycosylated linker. The fungus also produces a truncated form without the starch-binding domain (GA2). The active site mutant Trp(52)-->Phe of both forms and the Asp(55)-->Val mutant of the GA1 form have been prepared and physicochemically characterised and compared to recombinant wild-type enzymes. The characterisation included substrate hydrolysis, inhibitor binding, denaturant stability, and thermal stability, and the consequences for the active site of glucoamylase are discussed. The circular dichroic (CD) spectra of the mutants were very similar to the wild-type enzymes, indicating that they have similar tertiary structures. The D55V GA1 mutant showed slower kinetics of hydrolysis of maltose and maltoheptaose with delta delta G(double dagger) congruent with 22 kJ mol(-1), whereas the binding of the strong inhibitor acarbose was greatly diminished by delta delta G degrees congruent with 52 kJ mol(-1). Both W52F mutant forms have almost the same stability as the wild-type enzyme, whereas the D55V GA1 mutant showed slight destabilisation both towards denaturant and heat (DSC). The difference between the CD unfolding curves recorded by near- and far-UV indicated that D55V GA1 unfolds through a molten globule intermediate.

Synthesis and glycosidase inhibitory activity of 5-thioglucopyranosylamines. Molecular modeling of complexes with glucoamylase

The synthesis of a series of 5-thio-D-glucopyranosylarylamines by reaction of 5-thio-D-glucopyranose pentaacetate with the corresponding arylamine and mercuric chloride catalyst is reported. The products were obtained as anomeric mixtures of the tetraacetates which can be separated and crystallized. The tetraacetates were deprotected to give alpha/beta mixtures of the parent compounds which were evaluated as inhibitors of the hydrolysis of maltose by glucoamylase G2 (GA). A transferred NOE NMR experiment with an alpha/beta mixture of 7 in the presence of GA showed that only the alpha isomer is bound by the enzyme. The Ki values, calculated on the basis of specific binding of the alpha isomers, are 0.47 mM for p-methoxy-N-phenyl-5-thio-D-glucopyranosylamine (7), 0.78 mM for N-phenyl-5-thio-D-glucopyranosylamine (8), 0.27 mM for p-nitro-N-phenyl-5-thio-D-glucopyranosylamine (9) and 0.87 mM for p-trifluoromethyl-N-phenyl-5-thio-D-glucopyranosylamine (10), and the K(m) values for the substrates maltose and p-nitrophenyl alpha-D-glucopyranoside are 1.2 and 3.7 mM, respectively. Methyl 4-amino-4-deoxy-4-N-(5-thio-alpha-D-glucopyranosyl)-alpha-D-glucopyrano side (11) is a competitive inhibitor of GA wild-type (Ki 4 microM) and the active site mutant Trp120-->Phe GA (Ki 0.12 mM). Compounds 7, 8, and 11 are also competitive inhibitors of alpha-glucosidase from brewers yeast, with Ki values of 1.05 mM, > 10 mM, and 0.5 mM, respectively. Molecular modeling of the inhibitors in the catalytic site of GA was used to probe the ligand-enzyme complementary interactions and to offer insight into the differences in inhibitory potencies of the ligands.

Mutational analysis of catalytic mechanism and specificity in amylolytic enzymes

Abstract Engineering of proteins and substrates in conjunction with enzyme kinetic, thermodynamic and X-ray crystallographic studies has provided new knowledge on the mechanism of substrate binding and catalysis of starch-hydrolases and related enzymes. Enzyme-substrate interactions at a distance from the site of catalysis will receive special attention to expand the insight into the structural basis of the diversity known to amylolytic enzymes. Examples are reported of mutants of glucoamylase from Aspergillus niger and mutants and hybrids of barley α-amylase isozymes with altered specificity. In addition site-directed mutagenesis of barley α-amylase isozyme hybrids probes side chains critical for association with barley α-amylase/subtilisin inhibitor.

Glucoamylase mutants with decreased Km-values for C-6 substituted isomaltosides

Abstract Glucoamylase can catalyze the hydrolysis of methyl 6 R - C -alkyl α-isomaltosides. A Trp120→Phe mutant altered in the +2 binding subsite had a K m of 0.089 mM for methyl 6 R - C -methyl-α-isomaltoside compared to a K m of 0.71 mM for the wild-type enzyme. This reflects an eight-fold lower K m for this substrate; however, the k cat for the mutant was decreased 200-fold compared with the wild-type glucoamylase. With increasing size of the substituent to 6 R - C- ethyl and -isopropyl, Trp120→Phe and wild-type glucoamylase have similar K m values, while k cat for the mutant increases 10- and 100-fold, respectively, approaching wild-type values. Perturbation of the structural integrity around the general acid catalyst Glu179, through elimination of the Trp120 NE1 hydrogen-bond to Glu179 OE2 in Trp120→Phe glucoamylase, seems to be counteracted by the larger C-6 substituents. The apparent complementarity between enzyme and substrate analogs emphasizes the favorable impact of hydrophobic forces in protein-carbohydrate interactions. Wild-type glucoamylase and Val181→Ala/Asn182→Ala/Gly183→Lys/Ser184→His, a quadruple mutant located beyond subsite +3, essentially maintain k cat in substrates with the three different 6 R - C substituents, while K m increases from 0.45 to 47.0 mM for methyl-6 R - C- isopropyl α-isomaltoside. Compared with the wild-type enzyme, the quadruple mutant has 1.5–3.7-fold improved specificity ( k cat / K m ) for the parent and 6 R-C- alkyl isomaltosides, but 10-fold lower activity for the α-(1→4)-linked maltose caused mainly by a low k cat . This mutation at distant subsites thus influences hydrolysis of disaccharides, corroborating the presence of long-range effects on the catalytic site.