Paul J. Braun

Preparation and characterization of proteolyzed forms of human α-thrombin

Abstract The kinetics of the tryptic digestion of human α-thrombin were studied. Based on the results of these studies a procedure for the preparation of highly purified, active human β-thrombin was developed. This β-thrombin contained less than 5% of other thrombin forms, was active towards tripeptidyl paranitroanilide substrates, but had lost more than 99% of its fibrinogen cleaving activity. Protein-chemical characterization of β-thrombin showed that it had been cleaved at a single site (Arg 73 -Asn 74 ) in the B-chain, in contrast to human β-thrombin obtained by autolysis, which is cleaved at both Arg-62 and Arg-73.

Porcine-pancreatic alpha amylase hydrolysis of substrates containing 6-deoxy-D-glucose and 6-deoxy-6-fluoro-D-glucose and the specificity of subsite binding.

Hydrolysis of 6-deoxyamylose and mono-6-deoxy-6-fluorocyclomaltoheptaose by porcine-pancreatic alpha amylase produces low-molecular-weight modified products, which have been analyzed by chemical and chromatographic techniques. Results for both substrates show that modified D-glucose and two isomers of modified maltoses are produced in the enzyme reaction. In addition, the formation of maltoses modified in the nonreducing residue is more favored than the formation of maltoses modified in the reducing residue. These results indicate that productive binding of 6-fluoro- and 6-deoxy-D-glucose residues is permitted at subsites 1 through 4 of the amylase-active site but that binding of these modified residues may be less favorable at subsite 3, the subsite at which catalytic attack occurs.

The effect of substrate modification on binding of porcine pancreatic alpha amylase: Hydrolysis of modified amylose containing d-allose residues☆

A modified amylose containing 10% of tritiated D-allose residues has been hydrolyzed by porcine pancreatic alpha amylase (PPA). This reaction produced a number of radioactive oligosaccharides of low molecular weight, including modified mono-, di-, and tri-saccharides, as well as larger products. Analysis of these products by chemical and enzymic methods identified D-allose, two isomers of modified maltose, and isomers of modified maltotriose. These results may be interpreted in terms of current PPA models to indicate that D-allose residues may be productively bound at all five subsites of the active site of the enzyme. The distribution of modified residues in these products, however, further suggests that productive binding of D-allose at the subsite where catalytic attack occurs (subsite 3) is less favorable than binding of D-glucose. These results are compared with results of a series of PPA substrates having modifications at C-3 and at other positions. Trends observed in enzyme hydrolysis of these modified substrates reflect factors that contribute to PPA catalysis, with respect to steric, electronic, and hydrogen-bonding interactions between enzyme and substrate.

The effect of substrate modification on porcine pancreatic α-amylase subsite binding: Hydrolysis of substrates containing 2-deoxy-d-glucose and 2-amino-2-deoxy-d-glucose

Modified alpha-D-(1----4)-glucans containing a small proportion of 14C-labeled 2-deoxy-D-glucose or 2-amino-2-deoxy-D-glucose were examined as substrates for porcine pancreatic alpha-amylase (PPA). Cyclomaltoheptaose containing single 2-deoxy-D-glucose residues, synthesized by incubation of 2-deoxyglucosylglycogen with cyclomaltodextrin glucanotransferase in the presence of Triton X-100, was hydrolyzed by PPA to produce 2-deoxy-D-glucose; two isomers of 2-deoxymaltose, and a mixture of modified maltotrioses. These results indicate that 2-deoxymaltose, and a mixture of modified maltotrioses. These results indicate that 2-deoxy-D-glucose may be productively bound at all five subsites of the PPA active site. Reaction kinetics and the distribution of products formed suggest, however, that productive binding of the modified residue does not occur readily at the point of catalytic attack (subsite 3) and that the preferred position of hydrolysis of modified substrates may be different from that of unmodified substrates. Results of PPA hydrolysis of glycogen containing [14C]-2-amino-2-deoxy-D-glucose showed that a modified trisaccharide and a modified disaccharide were the smallest substituted products formed. Analysis of these products indicated that they did not contain modified residues at their reducing ends. Formation of the observed 2-amino-2-deoxy-maltooligosaccharides is consistent with a scheme where productive binding of 2-amino-2-deoxy-D-glucose is allowed at subsites 1, 2, 4, and 5, but not at subsite 3, the subsite at which hydrolysis occurs.