Identity and properties of the chloride effector binding site in hog pancreatic α-amylase

Lifshitz, Ruth; Levitzki, Alexander

doi:10.1021/bi00654a028

Cited by 41 publications

(24 citation statements)

References 15 publications

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“…When this lysine is selectively modified, C1 binding is abolished, but substrate binding is not affected. The persistence of a low basal activity following this modification suggests that C1-is a non-essential activator (Lifshitz and Levitzki 1976). The crystal structure of PAA indicates that the critical lysine may be Lys257, although Arg 195 and Arg 337 are also nearby (Buisson et al 1987).…”

Section: Ci-binding To Amino Acid Residuesmentioning

confidence: 98%

“…Chloride binding appears to trigger a small, localized conformational change which suppresses the exchange of 26 protons and increases the apparent affinity of the enzyme for Ca 2÷ by 240-fold. In a later study, Lifshitz and Levitzki (1976) determined that there is one CI binding site per molecule, a site which probably consists of a lysine epsilon-amino group with a pK~ = 9.1. Calcium binding near this residue was suggested to explain the low pKa.…”

Section: Ci-binding To Amino Acid Residuesmentioning

confidence: 99%

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Chloride binding proteins: mechanistic implications for the oxygen-evolving complex of Photosystem II

Coleman

1990

Photosynth Res

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Chloride plays a key role in activating the photosynethetic oxygen-evolving complex (OEC) of Photosystem II, but the OEC is only one of many enzymes affected by this anion. Some of the mechanistic features of Cl(-) involvement in water-splitting resemble those of other proteins whose structure and chemistry are known in detail. An overview of the similarities and differences between these Cl(-)-binding systems is presented.

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Section: Ci-binding To Amino Acid Residuesmentioning

confidence: 98%

Section: Ci-binding To Amino Acid Residuesmentioning

confidence: 99%

Chloride binding proteins: mechanistic implications for the oxygen-evolving complex of Photosystem II

Coleman

1990

Photosynth Res

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“…Another important ion binding site in the animal cy-amylases is that for chloride, which acts as an allosteric activator (Levitzki & Steer, 1974;Lifshitz & Levitzki, 1976 in Figure4 and Kinemage 4. Reference to Figure 5 shows that two of the amino acids involved are conserved in the fungal enzymes, whereas the third, Arg 337, is replaced by an isoleucine.…”

Section: Homology Of Ion Binding and Active Site Residuesmentioning

confidence: 99%

The structure of human pancreaticα-amylase at 1.8 Å resolution and comparisons with related enzymes

1995

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The structure of human pancreatic a-amylase has been determined to 1.8 A resolution using X-ray diffraction techniques. This enzyme is found to be composed of three structural domains. The largest is Domain A (residues 1-99, 169-404), which forms a central eight-stranded parallel @-barrel, to one end of which are located the active site residues Asp 197, Glu 233, and Asp 300. Also found in this vicinity is a bound chloride ion that forms ligand interactions to Arg 195, Asn 298, and Arg 337. Domain B is the smallest (residues 100-168) and serves to form a calcium binding site against the wall of the @-barrel of Domain A. Protein groups making ligand interactions to this calcium include Asn 100, Arg 158, Asp 167, and His 201. Domain C (residues 405-496) is made up of antiparallel @-structure and is only loosely associated with Domains A and B. It is notable that the N-terminal glutamine residue of human pancreatic a-amylase undergoes a posttranslational modification to form a stable pyrrolidone derivative that may provide protection against other digestive enzymes. Structure-based comparisons of human pancreatic a-amylase with functionally related enzymes serve to emphasize three points. Firstly, despite this approach facilitating primary sequence alignments with respect to the numerous insertions and deletions present, overall there is only -15% sequence homology between the mammalian and fungal a-amylases. Secondly, in contrast, these same studies indicate that significant structural homology is present and of the order of -70%. Thirdly, the positioning of Domain C can vary considerably between a-amylases. In terms of the more closely related porcine enzyme, there are four regions of polypeptide chain (residues 237-250, 304-310, 346-354, and 458-461) with significantly different conformations from those in human pancreatic a-amylase. At least two of these could play a role in observed differential substrate and cleavage pattern specificities between these enzymes. Similarly, amino acid differences between human pancreatic and salivary a-amylases have been localized and a number of these occur in the vicinity of the active site.Keywords: amylase; crystallography; enzymology; glycogen; pancreatic; sequences; starch; structure a-Amylases catalyze the hydrolysis of a-1,4 glucan linkages in starch and are widely distributed in nature, being found in bacteria, plants and animals. In humans, a-amylase is composed of 496 amino acids in a single polypeptide chain, which is encoded on chromosome 1 as part of a multigene family (Gumucio et al., 1988). These genes are regulated so that different isozymes are synthesized in either salivary glands or the pancreas. The salivary and pancreatic a-amylases are highly homologous in terms of primary sequence (Nishide et al., 1986) but d o exhibit somewhat different cleavage patterns (Minamiura, 1988). The functional differences observed undoubtedly arise from the 15 amino Reprint requests to: Gary D. Brayer, Department of Biochemistry and Molecular Biology, University of B...

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“…Binding of one chloride ion at a specific site induces the allosteric activation of ␣-amylase (20,21). Chloride activation has been demonstrated in mammalian pancreatic and salivary ␣-amylases (1) and in the structurally related bacterial enzyme from Alteromonas haloplanctis (22,23) whereas most microbial ␣-amylases seem chloride independent (24).…”

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confidence: 99%

Structural and Functional Aspects of Chloride Binding to Alteromonas haloplanctis α-Amylase

Feller

Bussy

Houssier

et al. 1996

Journal of Biological Chemistry

146

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Chloride is the allosteric effector of vertebrate pancreatic and salivary alpha-amylases and of the bacterial alpha-amylase from Alteromonas haloplanctis. Activation experiments of A. haloplanctis alpha-amylase by several monovalent anions show that a negative charge, not restricted to that of Cl-, is essential for the amylolytic reaction. Engineering of the chloride binding site reveals that a basic residue is an essential component of the site. The mutation K337R alters the Cl--binding properties, whereas the mutation K337Q produces an active, chloride-independent enzyme. Comparison of the Kd values for Cl- in three homologous alpha-amylases also indicates that the binding affinity is dependent on the chloride coordination mode by this basic residue. Analysis of substrate and chloride binding according to the allosteric kinetic model shows that the chloride effector is not involved in substrate binding. By contrast, the pH dependence of activity and experiments of chemical modifications and Ca2+ inhibition show that the chloride ion is responsible for the pKa shift of catalytic groups and interacts with active site carboxyl groups.

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Identity and properties of the chloride effector binding site in hog pancreatic α-amylase

Cited by 41 publications

References 15 publications

Chloride binding proteins: mechanistic implications for the oxygen-evolving complex of Photosystem II

Chloride binding proteins: mechanistic implications for the oxygen-evolving complex of Photosystem II

The structure of human pancreaticα-amylase at 1.8 Å resolution and comparisons with related enzymes

Structural and Functional Aspects of Chloride Binding to Alteromonas haloplanctis α-Amylase

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