Journal
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Volume 23, Issue 14, Pages -Publisher
MDPI
DOI: 10.3390/ijms23147739
Keywords
amylase; amylopectin; enzyme; galactomannan; maltase; NMR; polysaccharide; protein; starch; sucrase
Funding
- PharmaLectin, Inc.
- Bioxytran, Inc.
- National Science Foundation [BIR-961477]
- University of Minnesota Medical School
- Minnesota Medical Foundation
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This study reveals the molecular mechanism of action of PLG-007 (GM alpha) on enzyme hydrolysis of glucose polymers, demonstrating that GM alpha is the primary active component of PLG-007 and exerts its effects through inhibiting alpha-amylase-mediated hydrolysis of starch and amylopectin.
PLG-007 is a developmental therapeutic compound that has been clinically shown to reduce the magnitude of postprandial glucose excursions and has the potential to be an adjunct treatment for diabetes and inflammatory-related diseases. The present investigation is aimed at understanding the molecular mechanism of action of PLG-007 and its galactomannan (GM) components GM alpha and GM beta (in a 1:4 mass ratio, respectively) on enzyme (i.e., alpha-amylase, maltase, and lactase) hydrolysis of glucose polymers using colorimetric assays and C-13 HSQC NMR spectroscopy. The starch-iodine colorimetric assay indicated that GM alpha strongly inhibits alpha-amylase activity (similar to 16-fold more potent than GM beta) and thus is the primary active component in PLG-007. C-13 HSQC experiments, used to follow the alpha-amylase-mediated hydrolysis of starch and amylopectin, further demonstrate the alpha-amylase inhibitory effect of GM alpha via alpha-amylase-mediated hydrolysis of starch and amylopectin. Maltohexaose (MT6) was used to circumvent the relative kinetic complexity of starch/amylopectin degradation in Michaelis-Menten analyses. The V-max, K-M, and K-i parameters were determined using peak volume integrals from C-13 HSQC NMR spectra. In the presence of PLG-007 with alpha-amylase and MT6, the increase in K-M from 7.5 +/- 0.6 x 10(-3 )M (control) to 21 +/- 1.4 x 10(-3) M, with no significant change in V-max, indicates that PLG-007 is a competitive inhibitor of a-amylase. Using K-M values, K-i was estimated to be 2.1 +/- 0.9 x 10(-6) M; however, the microscopic K-i value of GM alpha is expected to be larger as the binding stoichiometry is likely to be greater than 1:1. Colorimetric assays also demonstrated that GM alpha is a competitive inhibitor of the enzymes maltase and lactase. Overall, this study provides insight as to how PLG-007 (GM alpha) is likely to function in vivo.
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