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PDBsum entry 1xh0

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Hydrolase PDB id
1xh0
Jmol
Contents
Protein chain
496 a.a.
Ligands
NAG
AAO
Waters ×280

References listed in PDB file
Key reference
Title Structural and mechanistic studies of chloride induced activation of human pancreatic alpha-Amylase.
Authors R.Maurus, A.Begum, H.H.Kuo, A.Racaza, S.Numao, C.Andersen, J.W.Tams, J.Vind, C.M.Overall, S.G.Withers, G.D.Brayer.
Ref. Protein Sci, 2005, 14, 743-755. [DOI no: 10.1110/ps.041079305]
PubMed id 15722449
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.
Figure 5.
Figure 5. Schematic representations of the hydrogen bond interactions formed within the chloride binding sites in wild-type HPA (and with bound acarbose) (A), N298S HPA with bound chloride (B), N298S HPA with both bound chloride and acarbose (C), N298S HPA in the absence of chloride (D), and N298S HPA in the absence of chloride, with acarbose bound (E). Note that in the N298S structure without bound chloride, the side chains of D300 and E233 are substantially shifted. For the N298S HPA-acarbose complex (in the absence of chloride), the side chain of E233 takes on two orientations that are approximately equally populated. Here conformation 1 is that found in the wild-type enzyme, while conformation 2 is that observed for the N298S variant without bound chloride and in the absence of acarbose. Bound water molecules are indicated by "Wat" and hydrogen bonds are designated by dashed lines.
Figure 6.
Figure 6. A schematic diagram summarizing the hydrogen bond interactions (dashed lines) to acarbose, when this inhibitor is bound in the active site of the N298S variant of HPA in the presence (A) and the absence (B) of chloride ion. The normal product of enzymatic inhibitor rearrangement is found in the presence of chloride. Surprisingly, an extended inhibitor product, with an additional glucose unit in binding subsite +3, is found in the absence of chloride (indicated by the dashed box). Active site cleft subsite binding locations have been indicated adjacent to each inhibitor sugar ring. Other prominent hydrogen bonding differences between these complexes occur in subsites -3 and +2. The water molecule believed to play a role in nucleophilic attack on the covalent intermediate generated during hydrolysis of substrates, has been indicated with an *.
The above figures are reprinted by permission from the Protein Society: Protein Sci (2005, 14, 743-755) copyright 2005.
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