Amino acid sequence of hog pancreatic α‐amylase isoenzyme I

Kluh, I.

doi:10.1016/0014-5793(81)80624-2

Cited by 55 publications

(22 citation statements)

References 11 publications

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“…spontaneously (Kluh, 1981;Bodansky & Martinez, 1983) and results in the loss of the terminal ND2 amide nitrogen group of the side chain of Gln 1 in the form of NH,. Figure 3 shows the conformation of the polypeptide chain in the vicinity of Gln 1 and Tyr 2, and the unusual N-terminal end structure formed.…”

Section: Resultsmentioning

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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Section: Resultsmentioning

confidence: 99%

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

1995

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“…Although there is no general sequence homology between this glucoamylase and known ttamylases (10,18,24,32,33), it was observed that a short stretch of polypeptide chain, preceding Trp(120) in glucoamylase and the Trp(83) in the Taka-amylase A (20) (from A. oryzae) was identical ( Figure 5). The Trp(83) is located in the active site cleft of the three-dimensional structure ofTaka-amylase A and model fitting studies with amylose have recently proposed that Trp(83) participates in binding of this substrate (20).…”

Section: Discussionmentioning

confidence: 99%

Identification of an essential tryptophanyl residue in the primary structure of glucoamylase G2 from aspergillus niger

Clarke

Svensson

1984

Carlsberg Res. Commun.

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Enzymatically active, N-bromosuccinimide oxidized glucoamylase G2 (EC 3.2.1.3) was prepared in the presence of the inhibitor acarbose and inactive, oxidized G2 with capacity to bind substrate was prepared in the presence of maltose. Four out of 15 tryptophanyl residues were oxidized in the first derivative and five in the latter. In order to identify this fifth and essential residue, tryptie fragments of the two G2 derivatives were isolated. The peptide fragment from the inactive G2 der/vative containing the additional oxindolealaniae residue was Tocated in HPLC chromatograms by UV-absorption measurements. Normal and second derivative UV-spectra, amino acid composition and NH_,-terminal sequence of the isolated fragment led to identification of Trp(120) as a residue essential for activity. A short homologous amino acid sequence precedes both this Trp(120) and the Trp(83) which is involved in substrate binding in Taka-amylase A (J. Biochem. 95, 697-702 (1984)).

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“…No homology was apparent between the primary structures of Gl and or-amylases described in the literature (14,21,31,37,46,47). Three fungal carbohydrases, a mycodextranase (36), a cellobiohydrolase (9,13), and the glucoamylase G1 (34,44), which all attack insoluble substrates, i.e.…”

Section: Discussionmentioning

confidence: 99%

The complete amino acid sequence of the glycoprotein, glucoamylase G1, from Aspergillus niger

Svensson

Larsen

Svendsen

et al. 1983

Carlsberg Res. Commun.

188

158

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The primary structure ofglucoamylase G1 (EC 3.2.1.3) from Aspergillus niger has been determined. Fragments of GI were obtained by cleavage with either cyanogen bromide, hydroxylamine, or S. aureus V8 protease. The resulting peptides were separated using ion exchange chromatography on DEAE-Sephacel, gel filtration, and affinity chromatography on Con A-Sepharose. Secondary fragments were generated by cleavage with either o-iodosobenzoic acid or BNPS-skatole as well as by digestion with S. aureus V8 protease, trypsin, and endoproteinase Lys-C. These peptides were purified by the procedures mentioned above and by reverse phase HPLC. The present fragments were amino acid sequenced and this permitted, in combination with the tryptic peptides (Carlsberg Res. Commun. 48, 517-527 (1983)), identification of 574 of the 614 amino acid residues in Gl. Sequencing of glucoamylase Gl cDNA, constructed from A. niger total mRNA, enabled deduction of the sequence of the remaining 40 amino acid residues localized to 6 short stretches. From the alignment of the fragments the complete primary structure of the enzyme was established. The amino acid sequence corresponds to a molecular weight of the polypeptide moiety of 65,424. Including both hexosamine and neutral carbohydrate contents the molecular weight of the present sample of G1 was calculated to be about 82,000.The majority of the carbohydrate of Gl is found in a highly glycosylated region of 70 amino acid residues which comprises about 35 O-glycosyl serine and threonine residues. This region ends approximately 100 residues from the C-terminus of the enzyme. Two N-glycosylated positions were found in the central part of the polypeptide chain. The molecule contains 9 half-cystine residues. No homology is apparent between the sequence of glycoamylase and various at-amylases.Abbreviations: BNPS-skatole = 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine; ca = citraconyl; Con A = concanavalin A; DFP = diisopropylfluorophosphate; DPCC = diphenylcarbamyl chloride; EDTA = ethylenediaminetetraacetic acid, disodium salt; Hepes = N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid; Nemac = N-ethylmorpholine acetate; HPLC = high pressure liquid chromatography; PTH = phenylthiohydantoin; SDS-PAGE = polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate; 2-pe = 2-pyridylethyl; G I designates the larger and G2 the smaller of the forms ofglucoamylase from A. niger (44).

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Amino acid sequence of hog pancreatic α‐amylase isoenzyme I

Cited by 55 publications

References 11 publications

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

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

Identification of an essential tryptophanyl residue in the primary structure of glucoamylase G2 from aspergillus niger

The complete amino acid sequence of the glycoprotein, glucoamylase G1, from Aspergillus niger

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