Structural and mechanistic studies of chloride induced activation of human pancreatic alpha-amylase.
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.
Maurus
Robert
R
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada V6T 1Z3.
Begum
Anjuman
A
Kuo
Hsin-Hen
HH
Racaza
Andrew
A
Numao
Shin
S
Andersen
Carsten
C
Tams
Jeppe W
JW
Vind
Jesper
J
Overall
Christopher M
CM
Withers
Stephen G
SG
Brayer
Gary D
GD
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
D002712:Chlorides; D000516:alpha-Amylases; D020909:Acarbose
Protein Sci. 1999 Mar;8(3):635-43
10091666
Nat Struct Biol. 1999 May;6(5):432-6
10331869
Structure. 1999 Sep 15;7(9):1079-88
10508777
Acc Chem Res. 2000 Jan;33(1):11-8
10639071
Biochemistry. 2000 Apr 25;39(16):4778-91
10769135
Biochemistry. 2000 Aug 8;39(31):9099-107
10924103
Gene. 2000 Jul 25;253(1):95-105
10925206
J Biochem. 2001 Mar;129(3):461-8
11226887
Biochemistry. 2002 Jan 8;41(1):215-25
11772019
Biochemistry. 2002 Apr 2;41(13):4492-502
11914097
Protein Sci. 2002 Jun;11(6):1435-41
12021442
Curr Opin Chem Biol. 2002 Oct;6(5):619-29
12413546
Acta Crystallogr D Biol Crystallogr. 1996 May 1;52(Pt 3):435-46
15299664
Methods Enzymol. 1997;277:505-24
18488322
Acta Crystallogr B. 1991 Aug 1;47 ( Pt 4):535-44
1930835
Acta Crystallogr A. 1991 Mar 1;47 ( Pt 2):110-9
2025413
Biochemistry. 1990 Jul 3;29(26):6244-9
2207069
Methods Enzymol. 1997;276:307-26
27754618
Biochim Biophys Acta. 1989 May 1;995(3):214-20
2784982
FEBS Lett. 1973 Apr 1;31(1):89-92
4710004
Eur J Biochem. 1974 Jan 3;41(1):171-80
4856205
J Biol Chem. 1995 Nov 10;270(45):26778-81
7592915
J Mol Biol. 1995 Mar 3;246(4):545-59
7877175
J Mol Biol. 1995 Mar 17;247(1):99-110
7897663
J Mol Biol. 1994 May 27;239(1):104-21
8196040
J Mol Graph. 1993 Jun;11(2):134-8, 127-8
8347566
Protein Sci. 1995 Sep;4(9):1730-42
8528071
J Biol Chem. 1996 Sep 27;271(39):23836-41
8798613
Biochemistry. 1997 Sep 9;36(36):10837-45
9283074
J Mol Biol. 1998 Mar 27;277(2):393-407
9514750
Protein Sci. 1998 Mar;7(3):564-72
9541387
Structure. 1998 Mar 15;6(3):281-92
9551551
Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905-21
9757107