J. Biochem, 1982, Vol. 92, No. 3 653-659
© 1982 Japanese Biochemical Society
research-article |
Kinetic Study on Chemical Modification of Taka-Amylase A
I. Location and Role of Tryptophan Residues
ko KITALaboratory of Biophysical Chemistry, College of Agriculture, University of Osaka Prefecture Sakai, Osaka 591
1. Five and four tryptophan residues in Taka-amylase A [EC 3.2.1.1 [EC] ] of A. oryzae (TAA) were modified with dimethyl(2-hydroxy-5-nitrobenzyl)-sulfonium bromide (K-IWS) in the absence and the presence of 15% maltose (substrate analog), respectively. Only one tryptophan residue was modified with dimethyl(2-methoxy-5-nitrobenzyl)-sulfonium bromide (K-IIWS) irrespective of the presence or absence of maltose.
Kinetic parameters (molecular activity, K0, Michaelis constant, Km, and inhibitor constant, K1) of the enzyme modified with K-IWS and K-IIWS were determined. The K0 value decreased with increase in the number of modified residues, but Km and K1 values and the type of inhibition were not altered by the modification.
2. The fluorescence quenching reaction of TAA with N-bromosuccinimide (NBS) proceeded in three phases. The second-order rate constants of the three phases were determined to be (4.3±0.5) × 105 M–1.S–1, (2.1 ±0.3) × 103 M–1.S–1 and (1.7 ±0.2) × 102 M–1.S–1, respectively. In the presence of maltose, the first phase was further separated into two phases with rate constants of (4.6±0.6)×105 M–1.S–1 and (6.9 ± 1.1) × 104 M–1.S–1, respectively.
On the basis of the results, it is estimated that five out of nine tryptophan residues are accessible to the solvent and among them, two tryptophan residues are substantially exposed: one is located in the maltose binding site near the catalytic site (its modification affects the catalytic function), and the other exists on the enzyme surface far from the active site.