Inhibition of a model protease--pyroglutamate aminopeptidase by a natural oligosaccharide gum from Hakea gibbosa.
The purpose of this study was to investigate the effect of the oligosaccharide gum from Hakea gibbosa on the activity of a model protease enzyme pyroglutamate aminopeptidase (5-oxoprolyl peptidase; EC 3.4.19.3) and to elucidate the mechanism responsible for the decreased activity. Enzyme kinetic studies were conducted at 37 degrees C in 100 mM potassium phosphate buffer with 10 mM EDTA, 5% (v/v) glycerol, and 5 mM DTT (pH 8) for 15 min and were performed both in the presence and absence of the gum. Enzymatic activity was determined by a colorimetric assay using the specific substrate L-pyroglutamic acid beta-napthylamide. The enzyme kinetics was studied at various substrate and gum concentrations. The velocity of the reaction was determined by the amount of the product (beta-napthylamine) liberated at each substrate and gum concentration. The Ks and Vmax of the enzyme in the absence of the gum were 24.40+/-2.14 microM and 502.95+/-28.90 nmoles x min(-1) x mg protein(-1), respectively. As the concentration of the gum was gradually increased from 0.1 to 2%, the value of the Vmax decreased from 318.94+/-21.46 to 158.83+/-24.51 nmoles x min(-1) x mg protein(-1) while Ks increased from 17.42+/-4.6 to 63.03+/-1.89 microM. The mechanism for the inhibition of the enzyme by Hakea appeared to be a mixed-linear type (a type of non-competitive inhibition) as suggested from Hanes-Woolf, Dixon and Cornish-Bowden plots. The turnover number, kcat, calculated for the enzyme also decreased from 14.09+/-0.81 to 4.45+/-0.69 min(-1) as the concentration of the inhibitor was incrementally increased from 0 to 2% (w/v). The K(i) and alphaK(i) calculated from Dixon and Cornish-Bowden plots were found to be 0.31+/-0.11% (w/v) and 1.33+/-0.42% (w/v), respectively. The natural gum from Hakea gibbosa was effective in non-competitively inhibiting the enzyme pyroglutamate aminopeptidase. Thus, the natural gum may be a promising additive not only for its sustained-release and mucoadhesive properties as shown previously, but also for its ability to slow the enzymatic degradation of therapeutic polypeptides incorporated in dosage forms.
Alur
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HH
Division of Pharmaceutical Sciences, School of Pharmacy, Room 211A, Katz Pharmacy Building, University of Missouri-Kansas City, 5005 Rockhill Road, Kansas City, MO 64110, USA.
Desai
R P
RP
Mitra
A K
AK
Johnston
T P
TP
D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009844:Oligosaccharides / Q000494:pharmacology*; D010944:Plants / Q000737:chemistry*; D011480:Protease Inhibitors / Q000494:pharmacology*; D011751:Pyroglutamyl-Peptidase I / Q000037:antagonists & inhibitors*
D009844:Oligosaccharides; D011480:Protease Inhibitors; D011751:Pyroglutamyl-Peptidase I