STUDIES ON STRUCTURE AND FUNCTION OF BARLEY MALT ALPHA-AMYLASE

Ziel

Alpha-amylases are starch degrading enzymes with important biotechnical applications. So far a structural model is available for a mammalian alpha-amylases and 2 microbial alpha-amylases. Dependent on the enzyme, different products are obtained. It is therefore relevant to determine a structure for a cereal alpha-amylase which characteristically produces larger oligodextrins. A multidisciplinary strategy was thus applied to improve the insight at a molecular level into structure/function relationship and structure/stability relationship of the dominant barley malt alpha-amylase 2 and its interaction with the endogenous inhibitor barley amylase/subtilisin inhibitor (BASI).

In order to explore the sugar protein interactions at the molecular level a number of nonhydrolysable substrate analogues specifically adapted to the alpha-amylase substrate structural requirement were prepared in the form of thioglucosides. Thus 4-thioglucoside, 4'-thiomaltose, 1-thio-delta-D-glucose and 1-thio-alpha-maltose were prepared and have subsequently been used for the synthesis of photolabile derivatives. Two maltotriose analogues were also synthesised. The synthesis of the 4,4'-dithio-alpha-maltotrioside 1 was based on our previously reported in situ selective S-deacetylation and activation procedure of pentaacetylated alpha-D-1-thioglucose. A strategy was applied to obtain spacer modified disaccharide which can mimic the trisaccharide 1 involving an alpha-D-1-thioglycopyranoside that carries a substituted butyl aglycon which can allow coupling to the reducing moiety. 14 analogues have been synthesised, 2 having diazirine spacers effective in irreversible inactivation of porcine pancreatic alpha-amylase on irradiation with long wave ultraviolet (UV) light at 350 nm. Higher homologues of the analogue shown in the illustration will be prepared for investigation of binding to barley alpha-amylase. Kinetic measurements using the pancreatic enzyme indicated that reactive groups can be ope rating at either end of the maltodextrin or within its chain, thus giving the means to selectively modified different parts of the extended substrate binding site. After preparing a great number of different reactive oligosaccharide derivatives, all mimicking more or less extended maltodextrins and measuring their affinities for pancreatic alpha-amylase, selected compounds were tested for the chemical modification of the enzyme binding area. The labelling turned out to be very efficient. Considering the different structures of reactive ligands and also taking in account the different yields of radiolabelling, depending on which substrate is used, we are confident that specific subsite labelling has been achieved.

The structure of the first alpha-amylase from a higher plant, the barley malt high pI isozyme form alpha-2-2 has been determined and a model constructed at 3 angstroms resolution. Techniques have been developed to synthesize nonhydrolyzable oligosaccharide analogues suitable for photoaffinity labelling of endo-alpha-glucanases. A complementary deoxyribonucleic acid yeast expression system for production of high yields of higher plant proteins has also been developed. It was discovered that carboxy terminal processing of the amylase took place in the aleurone cells catalyzed by 1 or more carboxypeptidases from malt and resulting in several forms of the protein.

Wissenschaftliches Gebiet

Programm/Programme

FP1-BAP - Multiannual research action programme (EEC) in the field of biotechnology (BAP), 1985-1989

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Finanzierungsplan

CSC - Cost-sharing contracts

Koordinator

Albert-Ludwigs-Universität Freiburg

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Heinrich-von-Stephan-Straße 25
79100 Freiburg
Deutschland

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