Substitutions of alanine for cysteine at a reactive thiol site and for lysine at a pyridoxal phosphate binding site of 1-aminocyclopropane-1-carboxylate deaminase.
1-Aminocyclopropane-1-carboxylate (ACC) deaminase catalyzes the cyclopropane ring fragmentation and deamination of ACC. Replacement of cysteine with alanine at a reactive thiol site, Cys-162, of ACC deaminase did not affect the enzyme activity, in spite of the previous result that modification of Cys-162 caused complete loss of the enzyme activity. Substitution of glycine or valine for the cysteine residue gave a higher Km for ACC without a significant change of the K0, indicating that changes of the amino acid side chain had structural effects on substrate binding. Replacement of lysine with alanine at the pyridoxal phosphate (PLP) binding site of the ACC deaminase caused a lower content of PLP and loss of detectable activity of ACC deamination. This mutant enzyme, K51A, showed absorption peaks at 330 nm and 405 nm. The peak at 405 nm was shifted to about 425 nm by the addition of ACC, D-, L-alanine, and D-, L-serine. The formation of aldimine complexes indicated by the spectral shift was reversible. It is suggested that lysine 51 affects the formation of holoenzyme and is important in catalysis.
Murakami
T
T
Faculty of Agriculture, Hokkaido University, Sapporo, Japan.
Kiuchi
M
M
Ito
H
H
Matsui
H
H
Honma
M
M
D000042:Absorption; D000409:Alanine / Q000737:chemistry*; D001483:Base Sequence; D001665:Binding Sites; D019755:Carbon-Carbon Lyases; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D008190:Lyases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010957:Plasmids; D011732:Pyridoxal Phosphate / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism; D014633:Valine / Q000737:chemistry
D013438:Sulfhydryl Compounds; D011732:Pyridoxal Phosphate; C033634:1-aminocyclopropane-1-carboxylate deaminase; D008190:Lyases; D019755:Carbon-Carbon Lyases; D014633:Valine; D008239:Lysine; D003545:Cysteine; D000409:Alanine