Site-directed mutagenesis of histidine 93, aspartic acid 180, glutamic acid 205, histidine 290, and aspartic acid 291 at the active site and tryptophan 279 at the raw starch binding site in barley alpha-amylase 1.

M Søgaard,A Kadziola,R Haser,B Svensson

doi:10.1016/s0021-9258(18)41554-2

Abstract

The pseudotetrasaccharide acarbose has high affinity for the active site (Ki,app = 1 microM) and low affinity for a secondary site (Kd = 2.3 mM) in barley alpha-amylase 1, distinguished by inhibition kinetics and spectral perturbation. Mutants of putative catalytic residues, D180N, E205Q, and D291N, are inactive and display low affinity for acarbose-Sepharose. H93N and H290N mutants, at invariant residues, have kcat/Km for p-nitrophenylmaltoheptaoside of 0.3 and 1.2% of wild-type. A corresponding 370- and 85-fold increased Ki,app for acarbose and a lack of shifts in pH activity profiles indicate that these histidines participate in transition state stabilization but not directly in catalysis. This finding agrees with H bonding to OH groups of the valienamine ring of acarbose in the three-dimensional structure. Loss of inhibition above pH 6 supports that acarbose is most potent in protonated form. The low affinity site contains Trp278 and Trp279, known to bind cyclomaltoheptaose. While the W279A mutant has 10-fold decreased affinity for starch granules, production of Trp278 mutants failed. The invariant Trp278 is perhaps critical for stability or folding in cereal alpha-amylases.

Highlights

= 1 b ~ a)nd low affinity for a secondary site (& = 2.3 mM) in barley a-amylase involves a @-glycosylenzyme intermediate (3, 4), as reported for pancreatic a-amylase incubatewdith maltotetraose at low temperature (5)
Glutathione, linked in a plasmid was propagated in fermentor in 10- or 100-liter (W279A) disulfide bond to approximately 30% of the rAMYl molecules (23), cultures (22,23).The mutantenzymes were purified from the culture was eliminated by 0.1 M dithiothreitol in 20 mM Tris-HCI, pH8.0, at liquid by affinity chromatography on CMH-Sepharose and analyzed room temperature followed by dialysis against 20 mM NaOAc, 1mM
The affinity for starch granules (Fig. 4) was lo-fold reduced in W279A, the Kd in the absence of CMH being 2.0 mg/ml compared with 0.2 mg/ml for wt rAMY1

Summary

The pseudotetrasaccharide acarbose has high affinityfor the activesite

A corresponding370- and 85-fold increasedKi,,,, for acarbose and a lack of shifts inpH activity profiles indicate that these histidines participate in transition state stabilization but not directly in catalysis This finding agrees with H bonding to OH groups of the catalysis may proceed via a substrate carboxonium ion electrostatically stabilized by carboxylate groups, as in hen egg white lysozyme (7, 8). Conserved Asp'&, GIu~~', anAd spzg7(Taka-amylase A numbering) comprise two catalytic groups presumably actmutation W278A/W279A, involved in CMH binding (19). It wasfound that Asp"", GIu~"~, anAd sp291areessential for activity; Hisg3and Hiszg0stabilize the transition state;TrpZ7' is important for adsorption onto granular starch; and Trp278 seems criticalfor folding and/or conformational stability

Strains and Plasmids

RESULTS

Acarbose hydroxyl group”

Findings

DISCUSSION