Abstract
A method for evaluating the association and dissociation rate constants of interaction between a lysozyme and its substrate analogue, an immobilized p-aminophenyl-tri-N-acetyl-β-chitotrioside, by means of surface plasmon resonance has been developed. Site-specific immobilization of p-aminophenyl-tri-N-acetyl-β-chitotrioside, which is a product of p-nitrophenyl-tri-N-acetyl-β-chitotrioside, on carboxymethyldextran linked to the surface of the cuvette of the instrument, IAsys, was carried out by catalysis with EDC/NHS. The kinetic parameters of the interaction between hen or human lysozyme and the immobilized substrate analogue indicated that a larger dissociation constant of the human lysozyme-immobilized substrate analogue complex depended on a smaller association rate constant. The kinetic parameters of the interaction between the immobilized substrate analogue and a mutant hen lysozyme, in which Arg14 and His15 are deleted, with higher activity than the wild type hen lysozyme were measured. It was suggested that the higher activity of the mutant lysozyme was due to faster removal of the substrate from the active site cleft and/or the formation of a stabler and better complex as to hydrolysis.
| Original language | English |
|---|---|
| Pages (from-to) | 712-716 |
| Number of pages | 5 |
| Journal | Journal of Biochemistry |
| Volume | 124 |
| Issue number | 4 |
| DOIs | |
| Publication status | Published - Jan 1 1998 |
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All Science Journal Classification (ASJC) codes
- Biochemistry
- Molecular Biology
Cite this
Kinetic measurement of the interaction between a lysozyme and its immobilized substrate analogue by means of surface plasmon resonance. / Ueda, Tadashi; Tsurumaru, Miyako; Imoto, Taiji.
In: Journal of Biochemistry, Vol. 124, No. 4, 01.01.1998, p. 712-716.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Kinetic measurement of the interaction between a lysozyme and its immobilized substrate analogue by means of surface plasmon resonance
AU - Ueda, Tadashi
AU - Tsurumaru, Miyako
AU - Imoto, Taiji
PY - 1998/1/1
Y1 - 1998/1/1
N2 - A method for evaluating the association and dissociation rate constants of interaction between a lysozyme and its substrate analogue, an immobilized p-aminophenyl-tri-N-acetyl-β-chitotrioside, by means of surface plasmon resonance has been developed. Site-specific immobilization of p-aminophenyl-tri-N-acetyl-β-chitotrioside, which is a product of p-nitrophenyl-tri-N-acetyl-β-chitotrioside, on carboxymethyldextran linked to the surface of the cuvette of the instrument, IAsys, was carried out by catalysis with EDC/NHS. The kinetic parameters of the interaction between hen or human lysozyme and the immobilized substrate analogue indicated that a larger dissociation constant of the human lysozyme-immobilized substrate analogue complex depended on a smaller association rate constant. The kinetic parameters of the interaction between the immobilized substrate analogue and a mutant hen lysozyme, in which Arg14 and His15 are deleted, with higher activity than the wild type hen lysozyme were measured. It was suggested that the higher activity of the mutant lysozyme was due to faster removal of the substrate from the active site cleft and/or the formation of a stabler and better complex as to hydrolysis.
AB - A method for evaluating the association and dissociation rate constants of interaction between a lysozyme and its substrate analogue, an immobilized p-aminophenyl-tri-N-acetyl-β-chitotrioside, by means of surface plasmon resonance has been developed. Site-specific immobilization of p-aminophenyl-tri-N-acetyl-β-chitotrioside, which is a product of p-nitrophenyl-tri-N-acetyl-β-chitotrioside, on carboxymethyldextran linked to the surface of the cuvette of the instrument, IAsys, was carried out by catalysis with EDC/NHS. The kinetic parameters of the interaction between hen or human lysozyme and the immobilized substrate analogue indicated that a larger dissociation constant of the human lysozyme-immobilized substrate analogue complex depended on a smaller association rate constant. The kinetic parameters of the interaction between the immobilized substrate analogue and a mutant hen lysozyme, in which Arg14 and His15 are deleted, with higher activity than the wild type hen lysozyme were measured. It was suggested that the higher activity of the mutant lysozyme was due to faster removal of the substrate from the active site cleft and/or the formation of a stabler and better complex as to hydrolysis.
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UR - http://www.scopus.com/inward/citedby.url?scp=0031790675&partnerID=8YFLogxK
U2 - 10.1093/oxfordjournals.jbchem.a022171
DO - 10.1093/oxfordjournals.jbchem.a022171
M3 - Article
C2 - 9756615
AN - SCOPUS:0031790675
VL - 124
SP - 712
EP - 716
JO - Journal of Biochemistry
JF - Journal of Biochemistry
SN - 0021-924X
IS - 4
ER -