Identification of the reactive sulfhydryl group of 1-aminocyclopropane-1-carboxylate deaminase.
1-Aminocyclopropane-1-carboxylate (ACC) deaminase, a pyridoxal phosphate enzyme that catalyzes cyclopropane ring-opening and deamination of ACC, formed a quinoid intermediate with D-alanine, as shown by the appearance of a 510-nm absorption band. The presence of D-alanine also stimulated the inactivation of ACC deaminase with iodoacetamide. The increase of absorbance at 510 nm and the stimulation of the enzyme inactivation were temperature-dependent with a critical point at around 20 degrees C, indicating a conformational change of the enzyme. To identify a reactive thiol group, this stimulated inactivation and an iodoacetamide derivative, N-(iodoacetamidoethyl)-1-aminonaphthalene-5- sulfonic acid were used. The residue that was modified by the specific reagent was monitored by absorbance at 350 nm through the digestion by lysylendopeptidase and the fractionation of peptides, and it was located at Cys-162 near the midpoint of the whole peptide chain of the ACC deaminase.
Honma
M
M
Faculty of Agriculture, Hokkaido University, Sapporo, Japan.
Kawai
J
J
Yamada
M
M
D000409:Alanine / Q000378:metabolism / Q000494:pharmacology; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D000598:Amino Acids, Cyclic; D019755:Carbon-Carbon Lyases; D003545:Cysteine / Q000737:chemistry*; D007460:Iodoacetamide / Q000494:pharmacology; D008190:Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009282:Naphthalenesulfonates / Q000737:chemistry; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D000596:Amino Acids; D000598:Amino Acids, Cyclic; D009282:Naphthalenesulfonates; C023863:1-aminocyclopropane-1-carboxylic acid; C006421:1,5-I-AEDANS; C033634:1-aminocyclopropane-1-carboxylate deaminase; D008190:Lyases; D019755:Carbon-Carbon Lyases; D003545:Cysteine; D000409:Alanine; D007460:Iodoacetamide