Yasunori Nitta

Subsite affinities of glucoamylase: Examination of the validity of the subsite theory

Abstract 1. 1. The dependence of Michaelis constant ( K m and the molecular activity ( k 0 ) on the degree of polymerization ( n ) of maltooligosaccharides for the hydrolysis catalyzed by glucoamylase of Rhizopus delemar (α-1,4:1,6-glucan 4:6-glucohydrolase, EC 3.2.1.3) was studied at pH 4.50 and 25 °C, for a range of n from 2 to 15.5. 2. 2. The results were analyzed to evaluate the subsite affinities ( A i ) and the intrinsic rate constant of hydrolysis of substrate linkage in a productive complex ( k int ) according to the theory formerly developed by one of the authors (Hiromi, K. (1970) Biochem. Biophys. Res. Commun. 40, 1–6), which assumed the independency of k int on n and the additivity of A i . 3. 3. The values of K m and k 0 calculated with the above determined values of A i and k int were in excellent agreement with the experimentally obtained ones for n = 2–7 , which confirmed the validity of the theory.

Synthesis of O-α-d-glucopyranosyl-(1→4)-O-α-d-xylopyranosyl-(1→4)-O-α-d-xylopyranosyl-(1→4)-d-glucopyranose as a substrate analogue of alpha amylase

Abstract The tetrasaccharide α- d -Glc p -(1→4)-α- d -Xyl p -(1→4)-α- d -Xyl p -(1→4)- d -Glc p ( 1 ) has been synthesized, as a substrate analogue of alpha amylase, by silver perchlorate-catalyzed glycosylation of benzyl 2,3,6-tri- O -benzyl-4- O -(2,3-di- O -benzyl-α- d -xylopyranosyl)-β- d -glucopyranoside ( 30 ) with 2,3-di- O -benzyl-4- O -(2,3,4,6-tetra- O -benzyl-α- d -glucopyranosyl)-α- d -xylopyranosyl chloride or by methyl triflate-promoted condensation of 30 with methyl 2,3-di- O -benzyl-4- O -(2,3,4,6-tetra- O -benzyl-α- d -glucopyranosyl)-1-thio-β- d -xylopyranoside, followed by removal of protecting groups of the resulting tetrasaccharide derivative 40 .

Kinetic Study of the Active Site Structure of β-Amylase from Bacillus cereus var. mycoides

The subsite affinities of the active site of β-amylase from Bacillus cereus var. mycoides were evaluated based on Hiromis theory, using (14)C-radiolabeled maltooligosaccharides as substrate. It was estimated that the active site consisted of six subsites, and all subsite affinities could be evaluated. The active site had a common subsite arrangement with those of β -amylases from soybean and wheat bran. The intrinsic breakdown rate constant of α-1,4 glucosidic linkage (kint) was five to seven times as large as those of the other enzymes.From the pH dependence of log[k0/Km], pK values of two functional ionizable groups were pK1 =4.0 and pK2 = 8.4. The pK values were 0.5-0.6 units for pK1 and 0.2-0.3 units for pK2 larger than those of the other enzymes. For the affinity-labeling of this enzyme by 2, 3 epoxypropyl α-D-glucopyranoside (α-EPG), the binding affinity of α-EPG was 1-1.6kcal/mol larger than those of the other β-amylases.

Maltal Binding Mechanism and a Role of the Mobile Loop of Soybean β-Amylase

The inhibition of hydration of maltal (α-D-glucopyranosyl-(l→4)-2-deoxy-D-glucal) catalyzed by soybean ²-amylase with 4-0-α-D-glucopyranosyl-(l→4)-l-deoxynojirimycin (GDN) was investigated at 25°C and at pH 5.4. As the concentrations of GDN used were comparable to that of the enzyme, Hendersons treatment was applied to this system. It was found that two maltal molecules bind to the enzyme according to a random mechanism and GDN inhibits the hydration of maltal competitively at subsites 1 and 2, and noncompetitively at the other site. On the basis of this result, it was inferred that the role of the mobile loop of this enzyme is to create a convenient catalytic environment for the hydration, and the closing of the active site by the mobile loop is induced by the binding of maltal.

Crystallization and preliminary X-ray crystallographic study of disproportionating enzyme from potato

Disproportionating enzyme (D-enzyme; EC 2.4.1.25) is a 59 kDa protein that belongs to the alpha-amylase family. D-enzyme catalyses intramolecular and intermolecular transglycosylation reactions of alpha-1,4 glucan. A crystal of the D-enzyme from potato was obtained by the hanging-drop vapour-diffusion method. Preliminary X-ray data showed that the crystal diffracts to 2.0 A resolution and belongs to space group C222(1), with unit-cell parameters a = 69.7, b = 120.3, c = 174.2 A.

Synthesis of the oligosaccharides α-d-Glcp(1→4)-d-Xylp, α-d-Xylp-(1→4)-d-Glcp, α-d-Glcp-(1→4)-α-d-Glcp-(1→4)-d-Xylp, α-d-Glcp-(1→4)-α-d-Xylp-(1→4)-d-Glcp, and α-d-Xylp-(1→4)-α-d-Glcp-(1→4)-d-Glcp

Abstract Syntheses are described of the disaccharides, α- d -Glc p -(1→4)- d -Xyl p -(1→4)- d -Glc p , and α- d -Xyl p -(1→4)- d -yl p , and the trisaccharides, α- d -Glc p -(1→4)-α- d -Glc p -(1→4)- d -Xyl p , α- d - Glc p (1→4)-α- d -Xyl p -(1→4)- d -Glc p , and α- d -Glc p -(1→4)- d -Glc p as analogues of glucoamylase and soybean beta-amylases substrates.

Kinetic study on maltal binding site of sweet potato β-amylase

Sweet potato β -amylase catalyzes two different kinds of reactions: hydrolysis of amylose, etc. and hydration of maltal (α-D-gluco- pyranosyl-(1 →4)-2-deoxy-D-glucal). To investigate whether both the reactions are catalyzed at the same catalytic site or not, an inhibition study on both the reactions and an affinity-labeling of Glul87 were done using 4-O- α -D-glucopyranosyl-(1→4)-l-deoxy- nojirimycin (GDN) as an inhibitor. As GDN showed competitive inhibition with the same Ki for these reactions, it was concluded that both the reactions occur at the same catalytic site.

pH-Dependence of Soybean β-Amylase-mediated Reactions

Soybean α-amylase mediates three kinds of reactions: (1) the hydrolysis of amylose, etc.; (2) an esterification of a functional carboxylate of G1u186 with 2, 3-epoxypropyl α-D-glucopyranoside (α-EPG); (3) a hydration of maltal. Thus, what the functional groups for each reaction are, and whether the catalytic sites are the same, are interesting questions. For hydration of maltal, we here studied the binding properties and the pH dependence of the rate parameter (pKm, k2). It was found that the binding of two maltal molecules to the enzyme is required for the hydration, and that only one protonated functional group with pK=6.75 is concerned with the catalysis, although two functional groups with pK=3.5 and pK=8.2 for both the hydrolysis of amylose and the esterification of G1u186 with a-EPG. Hehre et al. (J. Biol. Chem., 261, 2147(1986)) have proposed a possible mechanism for the hydration of maltal catalyzed by sweet potato β-amylase and assumed a functional carboxyl group (pK=3.7) for the hydrolysis of amylose as catalytic group. If this assumption is true in the case of soybean B-amylase, the pK of the functional carboxyl group (normally pK=3.5) must be raised to 6.75 as a result of the maltal binding to the enzyme. From inhibition study with 4-O-α-glucopyranosyl (1→4)-1-deoxynojirimycin as strong competitive inhibitor, it was presumed that three reactions are catalyzed at the same place in the active site.