Inhibition, activation, and stabilization of α-amylase family enzymes

Acarbose is a well-known inhibitor of α-glucosidases, α-amylases, cyclomaltodextrin glucanyltransferase (CGTase), and dextransucrase. Bacillus stearothermophilus maltogenic amylase (BSMA) was found to hydrolyze acarbose to give acarviosine-glucose plus glucose and to carry out a transglycosylation reaction to give isoacarbose. Acarviosine-glucose was a potent inhibitor of baker’s yeast α-glucosidase, 430-times better than acarbose; isoacarbose inhibited porcine pancreatic α-amylase (PPA) 15-times better than acarbose. Many other acceptors reacted with acarbose and BSMA to give a series of acarbose analogues modified with acarviosine-glucose attached primarily to the C-6-OH of the nonreducing-end of the acceptor. Two interesting products were acarviosine-glucose attached to cellobiose and lactose. These acarbose analogues were inhibitors for β-glucosidase and β-galactosidase, with KI values of 0.52 μM and 159 μM, where acarbose was not an inhibitor at all for these enzymes. Modification at the nonreducing-end of acarbose was accomplished by reacting acarbose with cyclomaltohexaose and CGTase to give maltohexaose and maltododecaose attached to the C-4-OH of acarbose (G6-Aca & G12-Aca). These analogues were very potent inhibitors of α-amylases. G6-Aca inhibited α-amylases from Aspergillus oryzae (AOA), Bacillus amyloliquefaciens (BAA), human saliva (HSA), and PPA with KI values of 37, 33, 14, and 7 nM, respectively, and G12-Aca had KI values of 81, 59, 18, and 11 nM, respectively, the most potent α-amylase inhibitors known. PPA lost activity exponentially over 2 hrs under optimum conditions. Addition of 0.02% (w/v) Triton X-100 gave 41% activation with stabilization. Seven polyethylene glycols (PEGs) at 0.02% with molecular weights of 400 to 8 kDa and two polyvinyl alcohols of 10 and 50 kDa also gave activation with stabilization. These additives were examined for 10 starch-degrading enzymes and found to be primarily more effective than Triton X-100. PEG 1.5 and 2 kDa gave the maximum degrees of activation for PPA, AOA, BAA, BLA, β-amylase, isoamylase, and CGTase, ranging from 20% to 77%. Triton X-100 gave maximum activation for HSA (45%) and pullulanase (27%). It is postulated that the enzymes have several tertiary structural forms that in solution are in dynamic equilibrium with each other. The additives that give maximum activation bind to the protein-enzymes to give a single, optimum structure that is fixed and gives the maximum activity with stabilization.