The Structural Biology Study Of The Interaction Of Human Starch Metabolizing Enzymes α-1,4Glycosidic Bond Related And Their Inhibitors

In human, a series of α-glucosidase enzymes, such as salivary amylase (HSΑ),pancreas amylase (HPΑ), maltase-glucoamylase (MGΑM) and sucrose glucosidase(SI) were involved in glucose production in the process of starch digestion.α-glucosidase inhibitor (ΑGI) could inhibit the generation of glucose result ininhibiting postprandial hyperglycemia which make AGI a very effective therapeuticdrug for type2diabetes. The study of the interaction between α-glucosidase inhibitorand the target α-glucosidase not only provides us insight into the mode of action ofthe enzyme molecules but also help us to develop new drugs against type2diabetes.First, human pancreatic amylase (HPΑ), maltase-glucoamylase N-terminaldomain (MGAM-N) and maltase-glucoamylase C-terminal domain (MGAM-C)which related to starch internal α-1,4glycosidic bond cleavages were cloned andexpressed in Pichia pastoris， named as HPA-GS115， MGAM-N-GS115，MGAM-C-GS115. In this work, recombinant human MGAM-C has been cloned forthe first time make it possible for us to study the mechanism of HPA, MGAM-N andMGAM-C at the same time. Different protein purification methods were used, such asglycogen precipitation, hydrophobic interaction chromatography, metal ion affinitychromatography, ion exchange chromatography and molecular exclusionchromatography to obtain high-purity HPA, MGAM-N and MGAM-C recombinantprotein. At last, the pure HPA catalytic activity was up to136.5U/mg, the pureMGAM-N and MGAM-C catalytic activity were5.15U/mg and20.23U/mg,respectively.Pure HPA, MGAM-N and MGAM-C recombinant proteins were used to studyits catalytic role in glucose metabolism pathway. The catalytic specificity, substratespecificity and different α-glucoseglycosidase inhibitors tolerance ability of HPA,MGAM-N and MGAM-C were studied. Provide us an overall profile of the inhibitoryability of these inhibitors.The result showed that DNJ was the most effective inhibitor against MGAM.The Kivalues for the two catalytic domains were1.41and2.04μM for MGAM-N and MGAM-C, respectively. Acarviostatins2-03and3-03（A2-03，A3-03）were the bestinhibitors against HPA with relatively high inhibitory activity against MGAM-C. TheA2-03and A3-03inhibition constants, Ki, for HPA were15and14.3nM, and thosefor MGAM-C were6.02and6.08μM, respectively. These results suggest thatMGAM-N and MGAM-C differ in their substrate specificities and inhibitor tolerancedespite their structural relationship.The high purity of the HPA was used as the material in the preparation of HPAcrystals. We obtained crystals of HPA in complexes with a series of acarviostatininhibitors (A1-03, A2-03, A3-03, and A4-03). Structural analyses showed that A1-03undergoes a series of hydrolysis and condensation reactions in the HPA active site,similar to acarbose, while A2-03, A3-03, and A4-03likely undergo only hydrolysisreactions leaves seven sugar rings in HPA active site. The final seven sugar ringproduct was best suited for occupying the full active site and shows the most efficientinhibition of HPA. The high resolution structures reported identify first time aninteraction between an inhibitor and subsite-4of the HPA active site, makes asignificant contribution to the inhibitory effect. Our results provide importantinformation for the design of new drugs for HPA target.The crystal structure of MGAM and MGAM/acarbose were obtained for the firsttime. Acarbose spans subsites from-1to+3of MGAM-C active site withnon-hydrolyzable N-linked bond occupying the catalytic center. Kinetic studiesshowed that MGAM-N has similar binding constants for substrates G2G6(theoligosaccharide substrates with27glucose rings referred to as G2G7) whileMGAM-C has a clear preference for G3G6substrates over the G2substratemaltose, indicating that major differences exist in the active sites of MGAM-N andMGAM-C. MGAM-N-Y299W，MGAM-C-Y1251W and MGAM-C-deltaS Pichiapastoris expression strains were acquired through Dpn1mediated site-directedmutagenesis technology. A single mutation of Trp1251to replace tyrosine (Y1251W)in MGAM-C imparts a novel catalytic ability to digest branched α-1,6-linkedoligosaccharides. The sequence of an extra21amino acids in MGAM-C connects toits longer substrates preference. A mutant with a deletion of these extra21aminoacids in MGAM-C (referred to as MGAM-C-deltaS) showed similar substrate specificity with MGAM-N. This research explains the different molecular mechanismof substrate specificity of MGAM-N, MGAM-C and SI-N to a certain extent.In this study, the Pichia pastoris expression system was used for the expressionof HPA, MGAM-N and MGAM-C recombinant protein. The high-purity HPAMGAM-N and MGAM-C protein were use to enzymatic properties studies as well asinhibitory studies. The crystal structure of HPA in complex with A1-03, A2-03A3-03,A4-03and the crystal structure of MGAM-C, MGAM-C/acarbose showed an overallview of the enzymes targeted at starch α-1,4glycosidic bond digestion. Thesefindings clarify how acarviostatins drugs interact with HPA, acarbose interact withMGAM-C, and will benefit the theoretical and applied hypoglycemic drugdevelopment targets for the HPA and MGAM.