Pál Elödi

Studies on the active center of pancreatic amylase

Summary1.The fact that β-cyclodextrin inhibits the hydrolysis of amylase suggests that β-cyclodextrin binds to the active center of pancreatic α-amylase EC 3.2.1.1.2.As shown by preparative ultracentrifugation, amylase binds specifically three moles of β-cyclodextrin per mole enzyme and the binding can be characterized by a single dissociation constant.3.The dissociation constant of amylase-β-cyclodextrin complex determined by kinetic methods is KI = 200 µm, which agrees well with the value KD = 140 µm, determined by preparative ultracentrifugation, and with the value KS = 220 µm arrived at by spectrophotometric titration.4.Solvent perturbation studies indicate that out of the three bound β-cyclodextrin molecules only one interacts with a tryptophyl side chain of amylase.

The role of the sulfhydryl groups in the stabilisation of the structure of the D-glyceraldehyde-3-phosphate dehydrogenase

Abstract The effect of the blocking of the SH groups on the spatial structure of the swine muscle D -glyceraldehyde-3-phosphate dehydrogenase was studied. The treatment with p -chloromercuribenzoate resulted in significant changes in the optical rotation and the intrinsic viscosity of the protein, the D -glyceraldehyde-3-phosphate dehydrogenase molecule being altered in the direction of denaturation. From these data it is assumed that the SH groups may play an important role in the stability of the secondary structure of the enzyme.

Structural investigations on dehydrogenases by means of difference spectrophotometry

Summary 1. The distribution and localization of tyrosine in glyceraldehyde-3-phosphate dehydrogenase ( D -glyceraldehyde 3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) and in lactate dehydrogenase ( L -lactate:NAD+ oxidoreductase, EC 1.1.1.27) were studied by difference spectrophotometry through spectrophotometric titration, solvent perturbation and iodination. 2. In both dehydrogenases the alkaline dissociation of some phenolic hydroxyl groups is anomalous. The apparent pK′s are 11.3 and 11.5, and the titrations are irreversible. 3. As measured by the solvent perturbation method, maxima of 45 and 50% of the tyrosyl residues may be located on the surfaces of the glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenases respectively. 4. About 65 and 80% of the tyrosyl groups are accessible to iodine in glycine buffer (pH 9.2). The remainder becomes available to iodine only after the proteins have been denatured with concentrated urea. 5. On the basis of the above findings the structural composition of these dehydrogenases is discussed.

Studies on the active center of pancreatic amylase: II. Small angle X-ray scattering investigations

Summary1.The radius of gyration of α-amylase is 26.9 Å, as found by means of small angle X-ray scattering. This value decreases by 0.5 Å if amylase binds three moles ofβ-cyclodextrin per mole enzyme.2.In case of partial saturation, the solution contains only saturated amylase-cyclodextrin complexes (amylase/cyclodextrin molar ratio 1:3) and amylase molecules free of cyclodextrin. The binding of β-cyclodextrin follows the all-or-none mechanism.3.The specifically bound β-cyclodextrin molecules are accommodated in a trough of the amylase molecule. The plane of bound cyclodextrin is perpendicular to the longitudinal axis of the trough. It is suggested that the helical substrate, amylose, binds in this trough, too.