An X-ray structure analysis of a crystal of pig pancreatic alpha-amylase (EC 3.2.1.1) that was soaked with acarbose (a pseudotetrasaccharide alpha-amylase inhibitor) showed electron density corresponding to five fully occupied subsites in the active site. The crystal structure was refined to an R-factor of 15.3%, with a root mean square deviation in bond distances of 0.015 A. The model includes all 496 residues of the enzyme, one calcium ion, one chloride ion, 393 water molecules, and five bound sugar rings. The pseudodisaccharide acarviosine that is the essential structural unit responsible for the activity of all inhibitors of the acarbose type was located at the catalytic center. The carboxylic oxygens of the catalytically competent residues Glu233 and Asp300 form hydrogen bonds with the "glycosidic" NH group of the acarviosine group. The third residue of the catalytic triad Asp197 is located on the opposite side of the inhibitor binding cleft with one of its carbonyl oxygens at a 3.3-A distance from the anomeric carbon C-1 of the inhibitor center. Binding of inhibitor induces structural changes at the active site of the enzyme. A loop region between residues 304 and 309 moves in toward the bound saccharide, the resulting maximal mainchain movement being 5 A for His305. The side chain of residue Asp300 rotates upon inhibitor binding and makes strong van der Waals contacts with the imidazole ring of His299. Four histidine residues (His101, His201, His299, and His305) are found to be hydrogen-bonded with the inhibitor. Many protein-inhibitor hydrogen bond interactions are observed in the complex structure, as is clear hydrophobic stacking of aromatic residues with the inhibitor surface. The chloride activator ion and structural calcium ion are hydrogen-bonded via their ligands and water molecules to the catalytic residues.

The crystal structure of porcine pancreatic alpha‐amylase (PPA) has been solved at 2.9 A resolution by X‐ray crystallographic methods. The enzyme contains three domains. The larger, in the N‐terminal part, consists of 330 amino acid residues. This central domain has the typical parallel‐stranded alpha‐beta barrel structure (alpha beta)8, already found in a number of other enzymes like triose phosphate isomerase and pyruvate kinase. The C‐terminal domain forms a distinct globular unit where the chain folds into an eight‐stranded antiparallel beta‐barrel. The third domain lies between a beta‐strand and a alpha‐helix of the central domain, in a position similar to those found for domain B in triose phosphate isomerase and pyruvate kinase. It is essentially composed of antiparallel beta‐sheets. The active site is located in a cleft within the N‐terminal central domain, at the carboxy‐end of the beta‐strands of the (alpha beta)8 barrel. Binding of various substrate analogues to the enzyme suggests that the amino acid residues involved in the catalytic reaction are a pair of aspartic acids. A number of other residues surround the substrate and seem to participate in its binding via hydrogen bonds and hydrophobic interactions. The ‘essential’ calcium ion has been located near the active site region and between two domains, each of them providing two calcium ligands. On the basis of sequence comparisons this calcium binding site is suggested to be a common structural feature of all alpha‐amylases. It represents a new type of calcium‐protein interaction pattern.(ABSTRACT TRUNCATED AT 250 WORDS)

The fitting of sequenced peptides to a high-resolution X-ray map of phosphoglycerate kinase has yielded the complete sequence and structure of the horse muscle enzyme. Metal ADP and ATP substrates are bound to one of the two widely separated domains in an environment that seems unsuitable for phosphoglycerate binding. The most plausible binding site for the phosphoglycerate substrate is on the other domain about 10 A from the ATP, which implies the possibility of a large scale hinge-bending of the domains to bring the two substrates together in a water-free environment for catalysis.

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. We propose that an increased resilience of the molecular surface and a less rigid protein core, with less interdomain interactions, are determining factors of the conformational flexibility that allows efficient enzyme catalysis in cold environments.

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