Carbonic anhydrase (CA) plays important roles in biological processes such as photosynthesis, respiration, secretion of HCO3 -, pH homeostasis and ion exchange.The proteins commonly contain a zinc ion in the active site for catalyzing the hydration of CO2 and vice versa.It is known that there are three classes of CA, designated α-, βand γ-CAs, depending on the amino acid sequence similarities.The α-class is different from others in the structural architecture.Furthermore, even in the α-class, the enzyme from unicellular green alga, Chlamydomonas reinhardtii (chCA) is unique in posttranslational modifications that it is glycosylated and spliced into two peptides.Such glycosylations are found in only mammalian CAs but they are not spliced.To reveal the structural details and the role of N-glycosylation, an X-ray analysis of chCA has been performed.chCA is a homodimeric protein, the two subunits being crystallographically independent.In each subunit, residues from Ser298 to Asn345 are spliced to separate into long and short peptides.The two subunits are, however, linked together by a disulfide bond.In the catalytic site, a zinc ion is bound to the three conserved His163, His165 and His182 in a tetrahedral configuration.A water molecule is trapped at the fourth position of the Zn atom.The electron density maps indicate that N-glycosylations occur at the three sites, Asn101, Asn135 and Asn297.This structure is the first example of CA attached to N-glycosides.chCA molecules are interacted to each other with a six-fold screw symmetry to form a long column.Furthermore, they are fused through the lateral interactions like a beehive.Each catalytic site is exposed to the central tunnel.It suggests that chCA in the crystalline state also catalyze the reaction.
A mechanistic study of the poorly understood pathway by which the inhibitor acarbose is enzymatically rearranged by human pancreatic α-amylase has been conducted by structurally examining the binding modes of the related inhibitors isoacarbose and acarviosine-glucose, and by novel kinetic measurements of all three inhibitors under conditions that demonstrate this rearrangement process. Unlike acarbose, isoacarbose has a unique terminal α-(1−6) linkage to glucose and is found to be resistant to enzymatic rearrangement. This terminal glucose unit is found to bind in the +3 subsite and for the first time reveals the interactions that occur in this part of the active site cleft with certainty. These results also suggest that the +3 binding subsite may be sufficiently flexible to bind the α-(1−6) branch points in polysaccharide substrates, and therefore may play a role in allowing efficient cleavage in the direct vicinity of such junctures. Also found to be resistant to enzymatic rearrangement was acarviosine-glucose, which has one fewer glucose unit than acarbose. Collectively, structural studies of all three inhibitors and the specific cleavage pattern of HPA make it possible to outline the simplest sequence of enzymatic reactions likely involved upon acarbose binding. Prominent features incorporated into the starting structure of acarbose to facilitate the synthesis of the final tightly bound pseudo-pentasaccharide product are the restricted availability of hydrolyzable bonds and the placement of the transition state-like acarviosine group. Additional "in situ" experiments designed to elongate and thereby optimize isoacarbose and acarviosine-glucose inhibition using the activated substrate αG3F demonstrate the feasibility of this approach and that the principles outlined for acarbose rearrangement can be used to predict the final products that were obtained.
A mechanistic study of the essential allosteric activation of human pancreatic α-amylase by chloride ion has been conducted by exploring a wide range of anion substitutions through kinetic and structural experiments. Surprisingly, kinetic studies indicate that the majority of these alternative anions can induce some level of enzymatic activity despite very different atomic geometries, sizes, and polyatomic natures. These data and subsequent structural studies attest to the remarkable plasticity of the chloride binding site, even though earlier structural studies of wild-type human pancreatic α-amylase suggested this site would likely be restricted to chloride binding. Notably, no apparent relationship is observed between anion binding affinity and relative activity, emphasizing the complexity of the relationship between chloride binding parameters and the activation mechanism that facilitates catalysis. Of the anions studied, particularly intriguing in terms of observed trends in substrate kinetics and their novel atomic compositions were the nitrite, nitrate, and azide anions, the latter of which was found to enhance the relative activity of human pancreatic α-amylase by nearly 5-fold. Structural studies have provided considerable insight into the nature of the interactions formed in the chloride binding site by the nitrite and nitrate anions. To probe the role such interactions play in allosteric activation, further structural analyses were conducted in the presence of acarbose, which served as a sensitive reporter molecule of the catalytic ability of these modified enzymes to carry out its expected rearrangement by human pancreatic α-amylase. These studies show that the largest anion of this group, nitrate, can comfortably fit in the chloride binding pocket, making all the necessary hydrogen bonds. Further, this anion has nearly the same ability to activate human pancreatic α-amylase and leads to the production of the same acarbose product. In contrast, while nitrite considerably boosts the relative activity of human pancreatic α-amylase, its presence leads to changes in the electrostatic environment and active site conformations that substantially modify catalytic parameters and produce a novel acarbose rearrangement product. In particular, nitrite-substituted human pancreatic α-amylase demonstrates the unique ability to cleave acarbose into its acarviosine and maltose parts and carry out a previously unseen product elongation. In a completely unexpected turn of events, structural studies show that in azide-bound human pancreatic α-amylase, the normally resident chloride ion is retained in its binding site and an azide anion is found bound in an embedded side pocket in the substrate binding cleft. These results clearly indicate that azide enzymatic activation occurs via a mechanism distinct from that of the nitrite and nitrate anions.
Small-scale baking ovens are built by the local workers and usually no scientific principles are followed to design and build of it. Modern scientific principles and procedures are followed to design and fabricate the baking ovens using low-cost materials. The major drawbacks of this type of traditional oven are: improper control of baking temperature; contamination of products with ashes during heating and excess cost for heating. This study aimed at modeling heat and mass transfer during baking, and evaluating the quality characteristics of bread and cake. The problems were identified by conducting a one-time cross-sectional survey, and the heat and mass transfer were modeled by designing and fabricating an improved oven. This model predicted the bread temperature and moisture content at 160, 170, 180, 190 and 200°C oven temperatures. The result indicated that increasing the oven temperature from 160 to 200°C increased the bread crust temperature from 101.58 to 158.69°C. However, the temperature and weight of bread increased gradually with increasing baking time up to 18-20 min and then started declining until it reached equilibrium after 30 min. The weight loss of bread increased with increasing bread temperature. The model predicted fairly accurate bread temperature and weight loss. It predicted 20 to 132°C against the observed 22 to 115°C during baking at 200°C oven temperature, 0 to 40% weight loss against 0 to 49% observed weight loss. The developed improved oven required 25% less time for baking bread and cake compared to the traditional one. Loaves of bread baked in the improved oven had 27.4% lower moisture content, 660 cm3 higher volume, and 408 g lower crumb firmness value compared to the conventional baking process. The improved baking oven is, therefore, more efficient than traditional baking ovens in terms of heat and mass transfer, baking time and product quality.
2 Abstract: Toxicity evaluation of a pollutant will be highly useful in the final evaluation of designing 'safe level' or 'tolerable level' of pollution to the terrestrial biosphere and thus will have the way in establishing limits and levels of acceptability by biotic components. Keeping inview of this a static test was conducted to evaluate the toxicity of fluoride in silkworm Bombyx mori., L. As the 96 hr acute toxicity or short term test is the most commonly employed toxicity test, silkworms were treated orally with various doses of fluoride for 96 h and the percent mortality was recorded. The 96 h LD value was found to be 27.5 mg/kg b.w. 50
A new approach for the discovery and subsequent structural elucidation of oligosaccharide-based inhibitors of α-amylases based upon autoglucosylation of known α-glucosidase inhibitors is presented. This concept, highly analogous to what is hypothesized to occur with acarbose, is demonstrated with the known α-glucosidase inhibitor, d-gluconohydroximino-1,5-lactam. This was transformed from an inhibitor of human pancreatic α-amylase with a Ki value of 18 mm to a trisaccharide analogue with a Ki value of 25 μm. The three-dimensional structure of this complex was determined by x-ray crystallography and represents the first such structure determined with this class of inhibitors in any α-glycosidase. This approach to the discovery and structural analysis of amylase inhibitors should be generally applicable to other endoglucosidases and readily adaptable to a high throughput format. A new approach for the discovery and subsequent structural elucidation of oligosaccharide-based inhibitors of α-amylases based upon autoglucosylation of known α-glucosidase inhibitors is presented. This concept, highly analogous to what is hypothesized to occur with acarbose, is demonstrated with the known α-glucosidase inhibitor, d-gluconohydroximino-1,5-lactam. This was transformed from an inhibitor of human pancreatic α-amylase with a Ki value of 18 mm to a trisaccharide analogue with a Ki value of 25 μm. The three-dimensional structure of this complex was determined by x-ray crystallography and represents the first such structure determined with this class of inhibitors in any α-glycosidase. This approach to the discovery and structural analysis of amylase inhibitors should be generally applicable to other endoglucosidases and readily adaptable to a high throughput format. α-Amylases (EC 3.2.1.1) are endoglycosidases that hydrolyze α(1,4) glucosidic linkages with net retention of configuration at the anomeric center. From their primary structures, these enzymes have been classified into glycosyl hydrolase family 13 (1Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2623) Google Scholar, 2Henrissat B. Bairoch A. Biochem. J. 1993; 293: 781-788Crossref PubMed Scopus (1771) Google Scholar, 3Henrissat B. Callebaut I. Fabrega S. Lehn P. Mornon J.P. Davies G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7090-7094Crossref PubMed Scopus (516) Google Scholar), a family that also includes α-glucosidases and cyclodextrin glucanotransferases. Analysis of tertiary structures reveals that the catalytic domain of family 13 enzymes, especially the active site, is very well conserved (4Aghajari N. Feller G. Gerday C. Haser R. Protein Sci. 1998; 7: 564-572Crossref PubMed Scopus (161) Google Scholar, 5Brayer G.D. Luo Y. Withers S.G. Protein Sci. 1995; 4: 1730-1742Crossref PubMed Scopus (292) Google Scholar, 6Brzozowski A.M. Davies G.J. Biochemistry. 1997; 36: 10837-10845Crossref PubMed Scopus (196) Google Scholar, 7Machius M. Wiegand G. Huber R. J. Mol. Biol. 1995; 246: 545-559Crossref PubMed Scopus (308) Google Scholar, 8Kadziola A. Abe J. Svensson B. Haser R. J. Mol. Biol. 1994; 239: 104-121Crossref PubMed Scopus (230) Google Scholar, 9Uitdehaag J.C. Mosi R. Kalk K.H. van der Veen B.A. Dijkhuizen L. Withers S.G. Dijkstra B.W. Nat. Struct. Biol. 1999; 6: 432-436Crossref PubMed Scopus (368) Google Scholar, 10Ramasubbu N. Paloth V. Luo Y.G. Brayer G.D. Levine M.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1996; 52: 435-446Crossref PubMed Scopus (227) Google Scholar). Indeed, a number of studies have shown that members of this family of glycosidases utilize a common double displacement catalytic mechanism (9Uitdehaag J.C. Mosi R. Kalk K.H. van der Veen B.A. Dijkhuizen L. Withers S.G. Dijkstra B.W. Nat. Struct. Biol. 1999; 6: 432-436Crossref PubMed Scopus (368) Google Scholar, 11McCarter J.D. Withers S.G. J. Biol. Chem. 1996; 271: 6889-6894Abstract Full Text PDF PubMed Scopus (121) Google Scholar, 12Rydberg E.H. Li C. Maurus R. Overall C.M. Brayer G.D. Withers S.G. Biochemistry. 2002; 41: 4492-4502Crossref PubMed Scopus (103) Google Scholar, 13Tao B.Y. Reilly P.J. Robyt J.F. Biochim. Biophys. Acta. 1989; 995: 214-220Crossref PubMed Scopus (60) Google Scholar), in which a glycosyl-enzyme intermediate is formed and hydrolyzed with acid/base catalysis via oxocarbenium ion-like transition states (14Sinnott M.L. Chem. Rev. 1990; 90: 1171-1202Crossref Scopus (1491) Google Scholar, 15Rye C.S. Withers S.G. Curr. Opin. Chem. Biol. 2000; 4: 573-580Crossref PubMed Scopus (435) Google Scholar, 16Zechel D.L. Withers S.G. Acc. Chem. Res. 2000; 33: 11-18Crossref PubMed Google Scholar). In humans, the pancreatic α-amylase (HPA) 1The abbreviations used are: HPA, human pancreatic α-amylase; CGTase, cyclodextrin glucanotransferase; CNP-G3, 2-chloro-4-nitrophenyl α-maltotrioside; G3F, α-maltotriosyl fluoride; G2-GHIL, maltosyl-α(1,4)-d-gluconohydroximino-1,5-lactam; GHIL, d-gluconohydroximino-1,5-lactam; MeG2F, 4′-O-methyl α-maltosyl fluoride; MeG2-GHIL, 4″-O-methyl-maltosyl-α(1,4)-d-gluconohydroximino-1,5-lactam; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. is responsible for cleaving large malto-oligosaccharides to smaller oligosaccharides, which are then substrates for intestinal α-glucosidases (Fig. 1). This digestion process is important for glucose absorption from the intestine to the blood, and in principle, control of HPA activity can be used as a means of controlling blood glucose levels. In fact, HPA activity has been correlated to post-prandial blood glucose levels (17Jenkins D.J. Taylor R.H. Goff D.V. Fielden H. Misiewicz J.J. Sarson D.L. Bloom S.R. Alberti K.G. Diabetes. 1981; 30: 951-954Crossref PubMed Google Scholar, 18Meyer B.H. Muller F.O. Kruger J.B. Clur B.K. Grigoleit H.G. S. Afr. Med. J. 1984; 66: 222-223PubMed Google Scholar, 19Taylor R.H. Jenkins D.J. Barker H.M. Fielden H. Goff D.V. Misiewicz J.J. Lee D.A. Allen H.B. MacDonald G. Wallrabe H. Diabetes Care. 1982; 5: 92-96Crossref PubMed Scopus (33) Google Scholar), and inhibitors of α-amylase have been successfully used in the treatment of diseases such as diabetes or obesity where control of the blood glucose level is essential (20Bailey C.J. Chem. Ind. 1998; : 53-57Google Scholar). Arguably, the most studied inhibitor of α-amylase is the naturally occurring and commercially available drug, acarbose (Fig. 2a), which has a Ki value in the low nanomolar range. This pseudo-tetrasaccharide is composed of a valienamine (unsaturated cyclitol) linked “α(1,4)” by an amine linkage to 6″-deoxy-maltotriose. The valienamine portion mimics the flattened sugar ring of the oxocarbenium ion-like transition state, whereas the exocyclic nitrogen places a proton acceptor in a position to interact with the acid/base catalyst at the active site. Indeed, with another family 13 enzyme, the CGTase from Bacillus circulans, this compound has been demonstrated to be a transition state analogue (21Mosi R. Sham H. Uitdehaag J.C.M. Ruiterkamp R. Dijkstra B.W. Withers S.G. Biochemistry. 1998; 37: 17192-17198Crossref PubMed Scopus (59) Google Scholar). As would be expected, structural studies of acarbose bound to HPA and other family 13 glycosidases have shown the valienamine moiety binding to the -1 subsite (22Brayer G.D. Sidhu G. Maurus R. Rydberg E.H. Braun C. Wang Y.L. Nguyen N.T. Overall C.M. Withers S.G. Biochemistry. 2000; 39: 4778-4791Crossref PubMed Scopus (198) Google Scholar), where sugar distortion takes place in the presumed catalytic mechanism. Interestingly, when acarbose is bound to HPA in this structure, it is modified by the enzyme through the addition of a maltosyl unit to the nonreducing end of the valienamine ring and the loss of a glucose moiety at the reducing end (Fig. 2a). The inhibitor therefore occupies all five subsites of HPA (–3 to +2). Similar, although not identical, modifications have also been seen in structural studies of other α-amylases in complexes with acarbose (6Brzozowski A.M. Davies G.J. Biochemistry. 1997; 36: 10837-10845Crossref PubMed Scopus (196) Google Scholar, 23Qian M.X. Haser R. Buisson G. Duee E. Payan F. Biochemistry. 1994; 33: 6284-6294Crossref PubMed Scopus (285) Google Scholar, 24Brzozowski A.M. Lawson D.M. Turkenburg J.P. Bisgaard-Frantzen H. Svendsen A. Borchert T.V. Dauter Z. Wilson K.S. Davies G.J. Biochemistry. 2000; 39: 9099-9107Crossref PubMed Scopus (124) Google Scholar). Because increased subsite occupancy is often associated with an increase in the overall binding affinity of an inhibitor, such a modification presumably results in a tighter binding molecule. To date, however, beyond the crystallographic data for the complex, there has been no direct evidence for such a product being formed in solution with α-amylase. Despite the considerable interest in α-amylases, there are relatively few other known inhibitors of this group of enzymes. From kinetic and structural studies, one would expect that the greatest transformation of the substrate on going from the ground state to the transition state will take place on the sugar occupying the -1 subsite. As such, the interactions at the -1 subsite should be particularly optimized for transition state binding, and therefore transition state mimics should bind particularly well at this site. Because the active sites of other family 13 α-glucosidases are thought to be structurally similar to that of HPA, it is likely that transition state analogues of such enzymes will interact similarly with the -1 subsite residues of α-amylase. Furthermore, it seems reasonable that the affinity of the inhibitor would correlate, in part, with the number of subsites with which the compound interacts. Therefore, an α-glucosidase inhibitor extended on the reducing and/or nonreducing end by a malto-oligosaccharide so as to occupy the other subsites should make a good inhibitor of α-amylases. In the past, the main difficulty in testing this hypothesis has been in the synthesis of such extended α-glucosidase inhibitors, although some work to this end has been carried out (25Takada M. Ogawa K. Saito S. Murata T. Usui T. J. Biochem. (Tokyo). 1998; 123: 508-515Crossref PubMed Scopus (8) Google Scholar, 26Takada M. Ogawa K. Murata T. Usui T. J. Carbohydr. Chem. 1999; 18: 149-163Crossref Scopus (2) Google Scholar, 27Uchida R. Nasu A. Tokutake S. Kasai K. Tobe K. Yamaji N. Chem. Pharm. Bull. 1999; 47: 187-193Crossref PubMed Scopus (15) Google Scholar, 28Yoon S.H. Robyt J.F. Carbohydr. Res. 2003; 338: 1969-1980Crossref PubMed Scopus (59) Google Scholar). In this paper, we describe a method to extend α-glucosidase inhibitors in situ, both in solution and within α-amylase crystals, by co-addition of an activated substrate. In doing so, we have been able to show that a potent α-glucosidase inhibitor, d-gluconohydroximino-1,5-lactam (GHIL; Fig. 2b) (29Hoos R. Vasella A. Rupitz K. Withers S.G. Carbohydr. Res. 1997; 298: 291-298Crossref Scopus (45) Google Scholar), which is normally a poor HPA inhibitor, is converted into a useful inhibitor of HPA. Using this method, it should be possible to assay a wide range of known α-glucosidase inhibitors using a facile kinetic screen for HPA inhibition by the elongated species and then determine the structure of the elongated species crystallographically. All of the chemicals and buffer salts were obtained from Sigma unless otherwise specified. HPA was purified according to literature procedures (30Rydberg E.H. Sidhu G. Vo H.C. Hewitt J. Cote H.C. Wang Y. Numao S. MacGillivray R.T. Overall C.M. Brayer G.D. Withers S.G. Protein Sci. 1999; 8: 635-643Crossref PubMed Scopus (59) Google Scholar). 2-Chloro-4-nitrophenyl α-maltotrioside (CNP-G3) was a generous gift from GelTex Inc. and can be purchased from Genzyme Inc. α-Maltotriosyl fluoride (G3F) (31Hayashi M. Hashimoto S. Noyori R. Chem. Lett. 1984; : 1747-1750Crossref Google Scholar) and GHIL (32Fleet G.W.J. Carpenter N.M. Petursson S. Ramsden N.G. Tetrahedron Lett. 1990; 31: 409-412Crossref Scopus (54) Google Scholar, 33Hoos R. Naughton A.B. Thiel W. Vasella A. Weber W. Rupitz K. Withers S.G. Helv. Chim. Acta. 1993; 76: 2666-2686Crossref Scopus (61) Google Scholar) were synthesized according to literature procedures. Syntheses of 2,4-dinitrophenyl α-maltotrioside and 4′-O-methyl α-maltosyl fluoride (MeG2F) will be published elsewhere. General—All of the studies were carried out at 30 °C in 50 mm sodium phosphate buffer, pH 6.9, containing 100 mm NaCl, unless otherwise described. Hydrolysis of CNP-G3 by HPA was monitored after the addition of enzyme by following the increase in absorbance at 400 nm using a Varian CARY 4000 spectrophotometer attached to a temperature control unit. The hydrolysis of G3F by HPA was monitored by following the increase in fluoride concentration upon the addition of enzyme using an ORION 96–04 combination fluoride electrode interfaced to a personal computer running the program LoggerPro (Vernier Software, Oregon). All analyses of the data were accomplished using the program Grafit 4.0.21 (34Leatherbarrow R.J. Grafit, 4.0.21 Ed. Erithacus Software Ltd., Staines, UK1998Google Scholar). Extension Kinetics and Product Analyses—Extension studies were accomplished by preincubating HPA with G3F (0.2 mm) and acarbose (180 nm), or G3F (0.4 mm) and GHIL (1 mm). After 1 h, the residual activity of HPA was measured by the addition of CNP-G3 and measuring the increase in absorbance at 400 nm. Control reactions were done in the absence of either the G3F or inhibitor. Product formation in the HPA-catalyzed reaction of G3F with GHIL was monitored by MALDI-TOF mass spectrometry analysis. G3F and GHIL were incubated with HPA (0.06 mg/ml) for 1 h at 30 °C in 5 mm sodium phosphate buffer, pH 6.9, containing 10 mm NaCl. A saturated solution of 2,5-dihydroxybenzoic acid in deionized water was used as the matrix. The sample and matrix solutions were mixed in a 1:20 ratio, and 1 μl of this sample was spotted on the target plate and dried in vacuo. The MALDI-TOF spectra were collected using a Voyager-DESTR (Applied Biosystems) mass spectrometer in reflectron mode with an acceleration voltage of 20 kV. Inhibition Kinetics—The Ki values and mode of inhibition were determined by measuring the rate of hydrolysis of either G3F (0.1225–2.45 mm) or CNP-G3 (0.5–2.0 mm) in the presence of a varying concentration of inhibitor (4–5 points). The data were fit using the program GraFit 4.0.21 (34Leatherbarrow R.J. Grafit, 4.0.21 Ed. Erithacus Software Ltd., Staines, UK1998Google Scholar) and plotted in the form of a Dixon plot (1/rate versus [inhibitor]) to allow visual inspection of the data. Enzymatic coupling of MeG2F (1.6 mg, 4.5 mmol) and GHIL (1.0 mg, 5.2 mmol) dissolved in 25 μl of 50 mm citrate buffer, pH 6.0, was accomplished by incubating the two compounds in the presence of CGTase (2.2 μl, 50 ng/ml) at 30 °C for 16 h. The reaction was monitored by MALDI-TOF mass spectrometry. After completion, the reaction mixture was freeze-dried, and the residue was purified by column chromatography using a water/acetonitrile solution as eluent (v/v, 1:4) to yield pure MeG2-GHIL in 70% yield (1.7 mg). Further characterization of the product was achieved after per-O-acetylation using acetic anhydride in pyridine overnight. The solvent was evaporated in vacuo, and the residue was dissolved in EtOAc. The solution was washed with aqueous HCl (1 m), saturated aqueous NaHCO3, and brine and then dried over MgSO4. NMR data: 1H NMR (CDCl3, 600 MHz): δ 5.81 (bs, 1 H, NH), 5.36 (m, 2 H, H-3, H-3′), 5.33 (m, 1 H, H-3″), 5.13 (d, 1 H, H-2), 5.06 (d, 1H,H-1″), 4.89 (d, 1 H, H-1′), 4.78 (dd, 1 H, H-2″), 4.74 (t, 1 H, H-4), 4.56 (dd, 1 H, H-2′), 4.46 (dd, 1 H, H-6a′), 4.28 (dd, 1 H, H-6a″), 4.06 (m, 3 H, H-6b′, H-5, H-6a), 3.93 (m, 2 H, H-6b, H-5′), 3.79 (dd, 1 H, H-6b″), 3.60 (t, 1 H, H-4′), 3.32 (m, 1 H, H-5″), 3.10 (s, 3 H, OCH3), 3.00 (t, 1 H, H-4″), 2.25–1.95 (10 s, 30 H, COCH3). Crystals of HPA were grown using conditions previously described (22Brayer G.D. Sidhu G. Maurus R. Rydberg E.H. Braun C. Wang Y.L. Nguyen N.T. Overall C.M. Withers S.G. Biochemistry. 2000; 39: 4778-4791Crossref PubMed Scopus (198) Google Scholar). The HPA/GHIL inhibitor complex was prepared for structural analysis by soaking a HPA crystal in a solution containing 100 mm GHIL for 24 h. A crystal of HPA complexed with both GHIL and G3F was prepared by first soaking in a solution containing 100 mm GHIL for 2 h followed by a 1-h soak in a solution containing 100 mm G3F. A similar approach was used in the formation of the complex with GHIL and MeG2F. Diffraction data for all complex crystals were collected on a Mar345 imaging plate area detector system at 100 K using copper Kα radiation supplied by a Rigaku RU300 rotating anode generator operating at 50 kV and 100 mA. Intensity data were integrated, scaled, and reduced to structure factor amplitudes, with the HKL suite of programs (35Otwinowski Z. Minor W. Method Enzymol. 1997; 276: 307-326Crossref Scopus (38567) Google Scholar). Data collection statistics are provided in Table I. The crystals of all of the complexes were isomorphous with that of HPA alone (22Brayer G.D. Sidhu G. Maurus R. Rydberg E.H. Braun C. Wang Y.L. Nguyen N.T. Overall C.M. Withers S.G. Biochemistry. 2000; 39: 4778-4791Crossref PubMed Scopus (198) Google Scholar), and as such, this structure was used as the starting refinement model for these complexes. Refinement was carried out with the CNS software program (36Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (8) Google Scholar). In these analyses, cycles of simulated annealing, positional, and thermal B refinements were alternated with manual model rebuilding with O (37Jones T.A. Zhou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13010) Google Scholar). In the initial stages of this process, the complete polypeptide chain of the refinement model was adjusted based on a composite annealed omit map (with 3% of the residues omitted per segment). Subsequently, the complete polypeptide chain was examined periodically with Fo - Fc, 2Fo - Fc and omit difference electron density maps. Following the refinement of the polypeptide chain of HPA, bound inhibitor molecules were positioned on the basis of Fo - Fc difference electron density maps. For both complexes, protein and inhibitor atoms were then jointly refined to obtain the best overall fit. In addition, an N-acetylglucosamine moiety bound to the side chain of Asn461 was included in the structural model and refined accordingly. At this point, solvent molecules were identified from a further Fo - Fc difference electron density map and included in the refinement model based on the hydrogen bonding potential to protein atoms and the refinement of a thermal factor of <75 Å2. In a final phase, the protein, inhibitor, and solvent molecules were jointly refined to convergence. The final refinement statistics obtained are detailed in Table I.Table IStructure determination statisticsGHIL/HPAGHIL/G3F/HPAGHIL/MeG2F/HPAData collection parametersSpace groupP212121P212121P212121Unit cell dimensionsa52.852.352.2b68.968.667.4c132.4131.5130.2No. of unique reflections35,24934,12527,904Mean I/αIaThe values in parentheses refer to the highest resolution shell (2.02-1.95 or 1.97-1.90 Å)24.1 (6.3)27.6 (11.1)15.0 (6.4)MultiplicityaThe values in parentheses refer to the highest resolution shell (2.02-1.95 or 1.97-1.90 Å)6.4 (3.4)9.8 (3.3)9.3 (2.1)Merging R-factor (%)aThe values in parentheses refer to the highest resolution shell (2.02-1.95 or 1.97-1.90 Å)6.2 (18.5)6.2 (18.0)4.8 (19.0)Maximum resolution (Å)1.951.901.95Structure refinement valuesNo. of reflections351553396627842Resolution range (Å)10.0-1.9510.0-1.9010.0-1.95Completeness within range (%)aThe values in parentheses refer to the highest resolution shell (2.02-1.95 or 1.97-1.90 Å)98.6 (96.9)89.9 (88.9)81.3 (80.2)No. of protein atoms394639463946No. of solvent atoms175161269Average B factors (Å2)Protein atoms20.420.219.8Solvent atoms29.329.824.6Final R-factor and R-free (%)b5% of the diffraction data was kept aside for the R-free16.7/19.817.5/19.518.3/22.7Structural stereochemistryRoot mean square deviationsBonds (Å)0.0070.0070.011Angles (°)1.31.31.4a The values in parentheses refer to the highest resolution shell (2.02-1.95 or 1.97-1.90 Å)b 5% of the diffraction data was kept aside for the R-free Open table in a new tab Kinetic Analyses—The concepts described herein first evolved out of our attempts to determine whether simple “monosaccharide” inhibitors of α-glucosidases would also function as useful inhibitors of HPA. As our best candidate, we investigated the potent α-glucosidase inhibitor GHIL (Ki = 2.9 μm with yeast α-glucosidase (29Hoos R. Vasella A. Rupitz K. Withers S.G. Carbohydr. Res. 1997; 298: 291-298Crossref Scopus (45) Google Scholar)). The assays were initially conducted with the substrate α-maltotriosyl fluoride (kcat = 200 s-1, Km = 0.5 mm) using a fluoride electrode. In this way, a Ki value of 1.8 mm was obtained. Although this inhibition is too weak to be therapeutically useful, it did suggest that this, and other such monosaccharides might be valuable inhibitors by providing a starting point for the design of extended and potentially more potent inhibitors that also occupy additional sugar binding subsites in the enzyme active site. Indeed, strong indications that elongation of these inhibitors with additional glucose residues might lead to substantial increases in affinity come from two findings. First, the kcat/Km values for oligosaccharide substrates were observed to significantly increase upon the addition of glucose moieties to the nonreducing end of a substrate. This points to substantial transition state stabilization from these additional sugar residues because the kcat/Km values increase from 98 s-1 mm-1 to 830 s-1 mm-1 on going from maltosyl fluoride to maltotriosyl fluoride (22Brayer G.D. Sidhu G. Maurus R. Rydberg E.H. Braun C. Wang Y.L. Nguyen N.T. Overall C.M. Withers S.G. Biochemistry. 2000; 39: 4778-4791Crossref PubMed Scopus (198) Google Scholar). Second, several studies have shown that oligosaccharide-based molecules are better inhibitors of the related porcine pancreatic α-amylase (25Takada M. Ogawa K. Saito S. Murata T. Usui T. J. Biochem. (Tokyo). 1998; 123: 508-515Crossref PubMed Scopus (8) Google Scholar, 26Takada M. Ogawa K. Murata T. Usui T. J. Carbohydr. Chem. 1999; 18: 149-163Crossref Scopus (2) Google Scholar, 27Uchida R. Nasu A. Tokutake S. Kasai K. Tobe K. Yamaji N. Chem. Pharm. Bull. 1999; 47: 187-193Crossref PubMed Scopus (15) Google Scholar). To more easily assay α-amylase inhibitors, a simple, chromogenic assay involving the commercially available substrate, CNP-G3 (kcat = 1.9 s-1, Km = 3.6 mm) was employed. To our surprise, using this compound as a substrate, a Ki value of 18 mm was found for GHIL, which is 10-fold higher than that measured using G3F as substrate. This unusual behavior merited further analysis because other data ensured that this was not an “artifact” of kinetic analysis and that there was indeed a change in the apparent affinity of HPA for GHIL depending on the substrate. Supportive evidence for such a change in inhibitory activity is evident when assaying for HPA activity with CNP-G3 at higher concentrations of GHIL, when a time-dependent decrease in rate was observed over a period of 20 min. No such time-dependent decrease was observed in the absence of inhibitor, eliminating substrate depletion as the cause of this phenomenon. Preincubation of HPA with GHIL prior to measurement of activity with CNP-G3 did not decrease initial rates, thereby eliminating both covalent inactivation and slow on rates as possible causes of this behavior. The most likely explanation for both the difference in Ki values and the time-dependent behavior is that during the kinetic analysis of GHIL with CNP-G3, slow transglycosylation of a maltotriosyl moiety to GHIL is occurring. This would yield an elongated version of GHIL that binds much more tightly than the original molecule, analogous to what is presumed to occur in crystals of HPA soaked with acarbose. However, because G3F is a much better substrate for HPA than is CNP-G3 (kcat/Km value is 1800 times greater than for CNP-G3), the maltotriosyl moiety will likely be transglycosylated to the inhibitor at a higher rate when working with G3F compared with CNP-G3. If this were the case, the “steady state” concentration of elongated species is quickly reached and at higher levels when using G3F than when using CNP-G3, thereby accounting for the 10-fold lower Ki value determined. For the more slowly reacting CNP-G3, formation of the steady state concentration of the elongated species occurs more slowly and is seen instead as a slow decrease in the catalytic activity over time. To probe this hypothesis further, HPA was preincubated with GHIL (0.4 mm) and G3F (1 mm) for 15 min prior to assay using CNP-G3 (1 mm). If an extended inhibitor is formed, then HPA should be inhibited under these conditions, even if the assay is done with CNP-G3. The rate determined under these conditions was ∼25% of the rate of a control reaction in which HPA was preincubated with G3F or GHIL alone, strongly suggesting that HPA is catalyzing a reaction between the inhibitor and substrate and that the product of this reaction is a better inhibitor of HPA (Fig. 3). A preincubation experiment was also carried out with acarbose (180 nm) in place of GHIL and similar results obtained, with rates significantly dropping compared with those for the control reaction in which no acarbose was present (data not shown). Because structural studies have shown acarbose to form an elongated product in the presence of HPA, this result provides strong support for the idea that elongated inhibitors are the principal inhibitory species in the kinetic analysis using G3F. Notably, as one might expect from this hypothesis, preincubation of HPA with G3F and glucose did not result in a significant decrease in the rate of hydrolysis of the substrate (Fig. 3). An attempt was made to quantitate the change in inhibitory activity by measuring an apparent Ki value for the elongated inhibitor species formed during preincubation. The experiments carried out involved incubation of varying concentrations of GHIL (0–0.6 mm) with a fixed concentration of G3F (0.4 mm) and HPA. After 1 h of incubation at 30 °C, the HPA activity was measured using 2,4-dinitrophenyl maltotrioside (0.9 mm). Rates measured under these conditions were then plotted in the form of a Dixon plot (assuming the complete conversion of GHIL to a homogeneous inhibitory species), and an apparent Ki value of 265 μm was extracted from the intersection between this line and the horizontal line corresponding to 1/Vmax (where Vmax is the maximal rate of 2,4-dinitrophenyl α-maltotrioside hydrolysis in the presence of 0.4 mm G3F) (data not shown). In comparison with the Ki value of 18 mm determined for unmodified GHIL (no G3F preincubation), this Ki value represents an approximate 100-fold increase in affinity as a consequence of preincubation. The greater inhibition observed under these conditions than when assayed directly with G3F suggests that longer reaction times and/or higher concentrations of reactants are required to build up inhibitory concentrations, even with G3F. The Ki value determined, however, represents a “worst case” estimate of the true Ki value for the inhibitor because it assumes all of the inhibitor is converted to the tight binding version, which is highly unlikely, and which mass spectrometric analysis (see below) reveals not to be the case. The true Ki value must therefore be much lower. Mass Spectrometric Analysis—Initial attempts to observe an elongated GHIL species formed under the preincubation conditions discussed above by using a MALDI-TOF mass spectrometer were unsuccessful. Only products of the normal hydrolysis and transglycosylation reactions of G3F were observed, possibly because the inhibitory species is being formed at concentrations well below the sensitivity level of the instrument. Concentrations of both G3F and GHIL were therefore raised such that G3F was present at 10 times its Km value, and GHIL was present at the Ki value of the unmodified species (18 mm). Analysis of such reaction mixtures by MALDI-TOF mass spectrometry revealed two additional products of m/z 539 and 702. These masses correspond to the sodium adducts of GHIL linked to a maltosyl or maltotriosyl moiety, respectively (Fig. 4), thereby demonstrating that HPA will catalyze the elongation of GHIL. Synthesis of an Extended GHIL for Further Kinetic Studies—To demonstrate that elongated GHIL is a better inhibitor of HPA, an extended version of GHIL was synthesized using a chemo-enzymatic approach (Fig. 5). To simplify both the enzymatic synthesis and subsequent kinetic analysis of the elongated species, a “blocked” version was made in which the 4″-hydroxyl group at the nonreducing end was methylated. Such an inhibitor should not undergo further elongation at the 4″-position and therefore should remain a stable species during kinetic analysis. Synthesis of this MeG2-GHIL was achieved by incubating MeG2F overnight with GHIL in the presence of CGTase from B. circulans, which has been shown previously to be highly effective in transferring sugar moieties to monosaccharide analogues (27Uchida R. Nasu A. Tokutake S. Kasai K. Tobe K. Yamaji N. Chem. Pharm. Bull. 1999; 47: 187-193Crossref PubMed Scopus (15) Google Scholar) without suffering the severe product inhibition that would be seen when using α-amylases. After the initial transglycosylation, the
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