Purification and characterization of a novel imidazole dipeptide synthase from the muscle of the Japanese eel Anguilla japonica.
We have purified a novel enzyme from eel white muscle which catalyzes the syntheses of imidazole dipeptides, such as carnosine (beta-alanyl-L-histidine), anserine (beta-alanyl-pi-methyl-L-histidine), and balenine (ophidine; beta-alanyl-tau-methyl-L-histidine), directly from their precursors. The enzyme was purified 1130-fold from eel muscle by a series of column chromatographies. Although eel muscle contains a large amount of carnosine and only trace amounts of anserine and balenine, the anserine synthesizing activity was by far the highest. From gel permeation chromatography, the molecular mass of the enzyme was calculated to be 275kDa. SDS-PAGE of the purified enzyme represented a band around 43kDa, suggesting that the native enzyme is a hexamer or heptamer. The optimal pH and temperature were around 9.5 and 60 degrees C, respectively. K(m) values for beta-alanine and pi-methyl-L-histidine were 44 and 89mM, respectively. The enzyme was greatly activated by Zn(2+) and inhibited by EDTA. The N-terminal amino acid sequence of 25 residues of the purified enzyme showed 52% amino acid identity to 38-62 residues of zebrafish haptoglobin precursor. The purified enzyme also exhibited hydrolytic activity against these imidazole dipeptides.
Tsubone
Shiori
S
Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan.
Yoshikawa
Naoko
N
Okada
Shigeru
S
Abe
Hiroki
H
D000595:Amino Acid Sequence; D000811:Anguilla; D000818:Animals; D000861:Anserine / Q000378:metabolism; D002336:Carnosine / Q000378:metabolism; D004150:Dipeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D004151:Dipeptides / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006242:Haptoglobins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007093:Imidazoles / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D010453:Peptide Synthases / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D029961:Zebrafish Proteins / Q000737:chemistry; D015032:Zinc / Q000378:metabolism
D004151:Dipeptides; D006242:Haptoglobins; D007093:Imidazoles; D029961:Zebrafish Proteins; C028119:N(beta)-alanyl-1-methyl-histidine; C029899:imidazole; D002336:Carnosine; D004492:Edetic Acid; D004150:Dipeptidases; D010453:Peptide Synthases; D000861:Anserine; D015032:Zinc