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

Abstract

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...

Stability of the Free and Bound Microstates of a Mobile Loop of α-Amylase Obtained from the Absolute Entropy and Free Energy

“…catalysis, 57 or assisting in the transition state, 66 or inducing a trap-release mechanism of substrate and products. 67 I.7.…”

Crystal structures of the psychrophilic α‐amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor

“…The wide range of known three-dimensional structures of a-amylases from Aspergillus oryzae (Matsuura et al, 1984;Swift et al, 1991), Aspergillus niger (Boel et al, 1990;Brady et al, 1991), porcine pancreas (Buisson et al, 1987;Qian et al, 1993;Larson et al. 1994), barley seeds (Kadziola et al, 1994), Bacillus lichenifomis (Machius et al, 1995), human pancreas (Brayer et al, 1995), and human salivary (Ramasubbu et al, 1996) constitute an ideal system for studies of adaptation of enzymes to extreme conditions on a molecular level. The three-dimensional structures obtained and described in this paper provide new insights into the mechanism of cold adaptation.…”

“…The structure of A. haloplanctis a-amylase cornplexed with Tris allows to understand the molecular basis of glycosidase inhibition by this cation which is a widely used compound in biochemistry. This bacterial a-amylase requires a chloride ion for its activity, a property previously considered as specific to mammalian a-amylases (Brayer et al, 1995). The structure of the chlorideloaded enzyme contributes to the understanding of the effect of this allosteric activator in the catalytic mechanism.…”

Knowledge‐based model of a glucosyltransferase from the oral bacterial group of mutans streptococci

“…Sequence alignment among amylases is also weak, although, to the contrary, 3D amylase structural alignment is substantial (Brayer et al, 1995). This led JaneEek (1996) to introduce the term "hidden homology" to describe this situation.…”

“…Seven solved crystal structures were aligned with the catalytic domain of GTF from Streptococcus downei: cyclodextrin glucanotransferase from Bacillus circulans (Klein & Schulz, 1991;Lawson et al, 1994); a-amylase from barley (Kadziola et al, 1994); porcine pancreatic a-amylase (Buisson et al, 1987;Larson et al, 1994); taka-amylase A from Aspergillus oryzae (Matsuura et al, 1984); oligo-1,6-glucosidase from Bacillus cereus (Kizaki et al, 1993); human pancreatic a-amylase (Brayer et al, 1995); and a-amylase from Aspergillus niger (Boel et al, 1990). The only blocks of interest in this analysis are those that show GTF/a-amylase sequence similarity.…”