An amylase inhibitor isolated from black beans (Phaseolus vulgaris) can completely inhibit porcine pancreatic α-amylase forming a 1:1 stoichiometric complex. The kinetic pattern of complex formation is pH dependent. At pH 5.5 it follows a first order reaction with rate constant of 0.029 min−1 and 0.017 min−1 at 37°C and equimolar inhibitor and enzyme concentration, respectively, of 10−8 M and 10−9 M. At pH 6.9 it is a second order reaction, with a rate constant of 0.25 × 106 M−1 min−1 at 37°C, with 4 × 10−8 M concentrations of enzyme and inhibitor. The dissociation constants of the enzymeinhibitor complex are 1.7 × 10−10 M at pH 5.5 and 4.4 × 10−9 M at pH 6.9, at 37°C. The kinetic data obtained at pH 5.5 suggested the formation of an initial reversible complex followed by a conformational change step. The complex can be dissociated either in acid pH (4.3) or at pH values higher than 6, 5 with partial recovery of the amylase activity.

Several amino acid residues important for the action of porcine pancreatic α-amylase on starch were modified using specific reagents: histidine groups by photooxidation with rose bengal or by diethylpyrocarbonate; cysteine by dithiobis nitrobenzoic acid; tryptophan by N-bromosuccimide and tyrosine by hydroxyl acetylation with N-acetylimidazole. These modifications, with the exception of cysteine, reduced the amylase activity but none of them alone was able to alter significantly the inhibition of the enzyme by a purified black bean (Phaseolus vulgaris) amylase inhibitor. Only by photooxidation that can modify several groups at the same time was the inhibitor action eliminated. The results suggested that the histidine of the amylase active site and tyrosine of the substrate binding site are not important for binding to the bean inhibitor. The terminal sugars of the amylase inhibitor were identified as mannose and xylose. Periodate oxidation of the carbohydrate moiety caused total loss of activity. The treatment of the inhibitor with α-mannosidase did not alter its inhibitor action on α-amylase.

Native chicken liver fructose-1,6-bisphosphatase (Fru-P2ase) can bind to blue dextranSepharose affinity column and is not displaced by its sugar-phosphate substrate; however; it is readily eluted by the inhibitor 5′-AMP. Treatment of Fru-P2ase with pyridoxal 5′-phosphate (pyridoxal-P) in the presence of the substrate, fructose 1,6-bisphosphate, followed by reduction with NaBH4 leads to the formation of active pyridoxal-P derivatives of the enzyme showing diminished sensitivity to AMP inhibitor. The modified enzyme does not bind to the affinity column. On the other hand, in the presence of AMP modification of Fru-P2ase with pyridoxal-P occurs at the catalytic site; this modification does not alter its binding behavior toward the dye ligand. Blue dextran can also protect Fru-P2ase against AMP inhibition, and it is a competitive desensitizer for the nucleotide ligand. The results establish that blue dextran binds specifically to the allosteric site of the enzyme, and that the structure of this site may resemble that of the dinucleotide fold in other enzymes. Like native Fru-P2ase, digestion of pyridoxal-P-Fru-P2ase (with regulatory properties altered) with subtilisin causes a severalfold increase in the catalytic activity measured at pH 9.2, without significant change in the activity at pH 7.5, and produces a peptide with 56 amino acids. The residual subunit, Mr ~ 30,000, was found to contain all of the incorporated pyridoxal-P.

Low molecular weight acid phosphatase (orthophosphoric monoester phosphophrydrolase (acid optimum), EC 3.1.3.2) from bovine brain is activated up to 4-fold by guanosine, guanine, adenine, adenosine, and 6-ethylmercapto-purine. Several pyrimidines and other purines were tested and did not show any activation effect. The rate enhancement induced by purines is uncompetitive and not caused by transphosphorylation to the activator. Using transphosphorylation to glycerel as a probe, it is proposed that the activator binds to one of the phosphorylated intermediates in the reaction pathway. These findings are discussed in terms of the catalytic mechanism of low molecular weight acid phosphatase.