title: Structural and mechanistic studies of chloride induced activation of human pancreatic alpha-amylase.
abstract: The mechanism of allosteric activation of alpha-amylase by chloride has been studied through structural and kinetic experiments focusing on the chloride-dependent N298S variant of human pancreatic alpha-amylase (HPA) and a chloride-independent TAKA-amylase. Kinetic analysis of the HPA variant clearly demonstrates the pronounced activating effect of chloride ion binding on reaction rates and its effect on the pH-dependence of catalysis. Structural alterations observed in the N298S variant upon chloride ion binding suggest that the chloride ion plays a variety of roles that serve to promote catalysis. One of these is having a strong influence on the positioning of the acid/base catalyst residue E233. Absence of chloride ion results in multiple conformations for this residue and unexpected enzymatic products. Chloride ion and N298 also appear to stabilize a helical region of polypeptide chain from which projects the flexible substrate binding loop unique to chloride-dependent alpha-amylases. This structural feature also serves to properly orient the catalytically essential residue D300. Comparative analyses show that the chloride-independent alpha-amylases compensate for the absence of bound chloride by substituting a hydrophobic core, altering the manner in which substrate interactions are made and shifting the placement of N298. These evolutionary differences presumably arise in response to alternative operating environments or the advantage gained in a particular product profile. Attempts to engineer chloride-dependence into the chloride-independent TAKA-amylase point out the complexity of this system, and the fact that a multitude of factors play a role in binding chloride ion in the chloride-dependent alpha-amylases.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada V6T 1Z3.
authors.forename: Robert
authors.initials: R
authors.lastname: Maurus
authors.forename: Anjuman
authors.initials: A
authors.lastname: Begum
authors.forename: Hsin-Hen
authors.initials: HH
authors.lastname: Kuo
authors.forename: Andrew
authors.initials: A
authors.lastname: Racaza
authors.forename: Shin
authors.initials: S
authors.lastname: Numao
authors.forename: Carsten
authors.initials: C
authors.lastname: Andersen
authors.forename: Jeppe W
authors.initials: JW
authors.lastname: Tams
authors.forename: Jesper
authors.initials: J
authors.lastname: Vind
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Overall
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Withers
authors.forename: Gary D
authors.initials: GD
authors.lastname: Brayer
mesh_terms: D020909:Acarbose / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001236:Aspergillus oryzae / Q000201:enzymology / Q000235:genetics; D002712:Chlorides / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D016296:Mutagenesis; D010179:Pancreas / Q000201:enzymology* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D002712:Chlorides; D000516:alpha-Amylases; D020909:Acarbose
references.citation: Protein Sci. 1999 Mar;8(3):635-43
references.pmid: 10091666
references.citation: Nat Struct Biol. 1999 May;6(5):432-6
references.pmid: 10331869
references.citation: Structure. 1999 Sep 15;7(9):1079-88
references.pmid: 10508777
references.citation: Acc Chem Res. 2000 Jan;33(1):11-8
references.pmid: 10639071
references.citation: Biochemistry. 2000 Apr 25;39(16):4778-91
references.pmid: 10769135
references.citation: Biochemistry. 2000 Aug 8;39(31):9099-107
references.pmid: 10924103
references.citation: Gene. 2000 Jul 25;253(1):95-105
references.pmid: 10925206
references.citation: J Biochem. 2001 Mar;129(3):461-8
references.pmid: 11226887
references.citation: Biochemistry. 2002 Jan 8;41(1):215-25
references.pmid: 11772019
references.citation: Biochemistry. 2002 Apr 2;41(13):4492-502
references.pmid: 11914097
references.citation: Protein Sci. 2002 Jun;11(6):1435-41
references.pmid: 12021442
references.citation: Curr Opin Chem Biol. 2002 Oct;6(5):619-29
references.pmid: 12413546
references.citation: Acta Crystallogr D Biol Crystallogr. 1996 May 1;52(Pt 3):435-46
references.pmid: 15299664
references.citation: Methods Enzymol. 1997;277:505-24
references.pmid: 18488322
references.citation: Acta Crystallogr B. 1991 Aug 1;47 ( Pt 4):535-44
references.pmid: 1930835
references.citation: Acta Crystallogr A. 1991 Mar 1;47 ( Pt 2):110-9
references.pmid: 2025413
references.citation: Biochemistry. 1990 Jul 3;29(26):6244-9
references.pmid: 2207069
references.citation: Methods Enzymol. 1997;276:307-26
references.pmid: 27754618
references.citation: Biochim Biophys Acta. 1989 May 1;995(3):214-20
references.pmid: 2784982
references.citation: FEBS Lett. 1973 Apr 1;31(1):89-92
references.pmid: 4710004
references.citation: Eur J Biochem. 1974 Jan 3;41(1):171-80
references.pmid: 4856205
references.citation: J Biol Chem. 1995 Nov 10;270(45):26778-81
references.pmid: 7592915
references.citation: J Mol Biol. 1995 Mar 3;246(4):545-59
references.pmid: 7877175
references.citation: J Mol Biol. 1995 Mar 17;247(1):99-110
references.pmid: 7897663
references.citation: J Mol Biol. 1994 May 27;239(1):104-21
references.pmid: 8196040
references.citation: J Mol Graph. 1993 Jun;11(2):134-8, 127-8
references.pmid: 8347566
references.citation: Protein Sci. 1995 Sep;4(9):1730-42
references.pmid: 8528071
references.citation: J Biol Chem. 1996 Sep 27;271(39):23836-41
references.pmid: 8798613
references.citation: Biochemistry. 1997 Sep 9;36(36):10837-45
references.pmid: 9283074
references.citation: J Mol Biol. 1998 Mar 27;277(2):393-407
references.pmid: 9514750
references.citation: Protein Sci. 1998 Mar;7(3):564-72
references.pmid: 9541387
references.citation: Structure. 1998 Mar 15;6(3):281-92
references.pmid: 9551551
references.citation: Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905-21
references.pmid: 9757107