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)

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The a-amylase secreted by the antarctic bacterium Alteromonas haloplanctis displays 66% amino acid sequence similarity with porcine pancreatic a-amylase. The psychrophilic a-amylase is however characterized by a sevenfold higher k,,, and k,,/K,,, values at 4°C and a lower conformational stability estimated as 10 kJ . mol-' with respect to the porcine enzyme. It is proposed that both properties arise from an increase in molecular flexibility required to compensate for the reduction of reaction rates at low temperatures. This is supported by the fast denaturation rates induced by temperature, urea or guanidinium chloride and by the shift towards low temperatures of the apparent optimal temperature of activity.When compared with the known three-dimensional structure of porcine pancreatic a-amylase, homology modelling of the psychrophilic a-amylase reveals several features which may be assumed to be responsible for a more flexible, heat-labile conformation: the lack of several surface salt bridges in the @/a)* domain, the reduction of the number of weakly polar interactions involving an aromatic side chain, a lower hydrophobicity associated with the increased flexibility index of amino acids forming the hydrophobic clusters and by substitutions of proline for alanine residues in loops connecting secondary structures. The weaker affinity of the enzyme for Ca2+ (Kd = 44 nM) and for C1F (Kd = 1.2 mM at 4°C) can result from single amino acid substitutions in the Ca2+-binding and CIF-binding sites and can also affect the compactness of a-amylase.

Two different crystal forms of pig pancrleatic a-amylase isoenzyme I1 (PPAII), free and complexed to a carbohydrate inhibitor (acarbose), have been compared together and to previously reported structures of PPAI. A crystal form obtained at 4"C, containing nearly 72% solvent, made it possible to obtain a new complex with acarbose, different from a previous one obtained at 20°C [Qian, M., Buisson, G., DuCe, E., Haser, H. & Payan, F. (1994) Biocrbemistry 33, 6284-62941. In the present form, six contiguous subsites of the enzyme active site are occupied by the carbohydrate ligand; the structural data indicate that the binding site is capable of holding more than the five glucose units of the scheme proposed through kinetic studies. A monosaccharide ring bridging two protein molecules related by the crystal packing is located on the surface, at a distaince of 2.0 nm from the reducing end of the inhibitor ligand; the symmetry-related glucose ring in the crystal lattice is found 1.5 nm away from the non-reducing end of the inhibitor ligand.

Camelids produce functional antibodies devoid of light chains and CH1 domains. The antigen-binding fragment of such heavy chain antibodies is therefore comprised in one single domain, the camelid heavy chain antibody VH (VHH). Here we report on the structures of three dromedary VHH domains in complex with porcine pancreatic ␣-amylase. Two VHHs bound outside the catalytic site and did not inhibit or inhibited only partially the amylase activity. The third one, AMD9, interacted with the active site crevice and was a strong amylase inhibitor (K i ‫؍‬ 10 nM). In contrast with complexes of other proteinaceous amylase inhibitors, amylase kept its native structure. The water-accessible surface areas of VHHs covered by amylase ranged between 850 and 1150 Å 2 , values similar to or even larger than those observed in the complexes between proteins and classical antibodies. These values could certainly be reached because a surprisingly high extent of framework residues are involved in the interactions of VHHs with amylase. The framework residues that participate in the antigen recognition represented 25-40% of the buried surface. The inhibitory interaction of AMD9 involved mainly its complementarity-determining region (CDR) 2 loop, whereas the CDR3 loop was small and certainly did not protrude as it does in cAb-Lys3, a VHH-inhibiting lysozyme. AMD9 inhibited amylase, although it was outside the direct reach of the catalytic residues; therefore it is to be expected that inhibiting VHHs might also be elicited against proteases. These results illustrate the versatility and efficiency of VHH domains as protein binders and enzyme inhibitors and are arguments in favor of their use as drugs against diabetes.The fundamental molecular recognition molecules of the humoral immune response are remarkably homogeneous throughout the vertebrate phylum. All immunoglobulins are multimers of heterodimeric chains where each heavy (H) 1 chain of four or five domains is linked by disulfide bridges to a light (L) chain of two domains (1). The antigen-binding part of the immunoglobulins is formed invariably by the N-terminal domains of both the H and L chains. These domains display a large sequence variation concentrated in three regions per domain, the CDRs.However, important deviations of this conserved immunoglobulin organization have been observed. In some immunoglobulin isotypes of camelids from the old world (camels and dromedaries) or from the new world (llamas and vicunas) the L chain is missing (2). Furthermore their H chain is devoid of the CH1 domain (3, 4) due to an unconventional splicing event during the mRNA maturation. The antigen-binding fragment of the heavy chain antibodies is therefore comprised in one single domain, the unique N-terminal variable domain referred to as VHH that replaces a four-domain Fab fragment in the immunoglobulin structure (5). This VHH domain is obtained after a DNA recombination between dedicated VHH germline gene segments with D and J minigenes. The dromedary VHH germline genes are quite diverse. They...

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Mammalian alpha-amylases catalyze the hydrolysis of alpha-linked glucose polymers according to a complex processive mechanism. We have determined the X-ray structures of porcine pancreatic alpha-amylase complexes with the smallest molecule of the trestatin family (acarviosine-glucose) which inhibits porcine pancreatic alpha-amylase and yet is not hydrolyzed by the enzyme. A structure analysis at 1.38 A resolution of this complex allowed for a clear identification of a genuine single hexasaccharide species composed of two alpha-1,4-linked original molecules bound to the active site of the enzyme. The structural results supported by mass spectrometry experiments provide evidence for an enzymatically catalyzed condensation reaction in the crystal.