ABSTRACT
Acetyl-CoA carboxylase (ACC) catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, a key metabolite in the fatty acid synthetic and oxidation pathways. The present study describes the steady-state kinetic analysis of a purified recombinant human form of the enzyme, namely ACC2, using a novel LC/MS/MS assay to directly measure malonyl-CoA formation. Four dimensional matrices, in which bicarbonate (HCO(3)(-)), ATP, acetyl-CoA, and citrate were varied, and global data fitting to appropriate steady-state equations were used to generate kinetic constants. Product inhibition studies support the notion that the enzyme proceeds through a hybrid (two-site) random Ter Ter mechanism, one that likely involves a two-step reaction at the biotin carboxylase domain. Citrate, a known activator of animal forms of ACC, activates both by increasing k(cat) and k(cat)/K(M) for ATP and acetyl-CoA.
Subject(s)
Acetyl-CoA Carboxylase/chemistry , Acetyl-CoA Carboxylase/antagonists & inhibitors , Chromatography, Liquid , Humans , Kinetics , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Tandem Mass SpectrometryABSTRACT
Advanced glycation end products (AGEs) accumulate with age and at an accelerated rate in diabetes. AGEs bind cell-surface receptors including the receptor for advanced glycation end products (RAGE). The dependence of RAGE binding on specific biochemical characteristics of AGEs is currently unknown. Using standardized procedures and a variety of AGE measures, the present study aimed to characterize the AGEs that bind to RAGE and their formation kinetics in vitro. To produce AGEs with varying RAGE binding affinity, bovine serum albumin (BSA) AGEs were prepared with 0.5M glucose, fructose, or ribose at times of incubation from 0 to 12 weeks or for up to 3 days with glycolaldehyde or glyoxylic acid. The AGE-BSAs were characterized for RAGE binding affinity, fluorescence, absorbance, carbonyl content, reactive free amine content, molecular weight, pentosidine content, and N-epsilon-carboxymethyl lysine content. Ribose-AGEs bound RAGE with high affinity within 1 week of incubation in contrast to glucose- and fructose-AGE, which required 12 and 6 weeks, respectively, to generate equivalent RAGE ligands (IC50=0.66, 0.93, and 1.7 microM, respectively). Over time, all of the measured AGE characteristics increased. However, only free amine content robustly correlated with RAGE binding affinity. In addition, detailed protocols for the generation of AGEs that reproducibly bind RAGE with high affinity were developed, which will allow for further study of the RAGE-AGE interaction.
Subject(s)
Acetaldehyde/analogs & derivatives , Arginine/analogs & derivatives , Glycation End Products, Advanced/metabolism , Lysine/analogs & derivatives , Receptors, Immunologic/metabolism , Acetaldehyde/chemistry , Acetaldehyde/metabolism , Amines/analysis , Arginine/analysis , Diabetes Mellitus/metabolism , Fructose/chemistry , Fructose/metabolism , Glucose/chemistry , Glucose/metabolism , Glycation End Products, Advanced/chemical synthesis , Glyoxylates/chemistry , Glyoxylates/metabolism , Humans , Ligands , Lysine/analysis , Lysine/chemistry , Lysine/metabolism , Membrane Proteins/metabolism , Molecular Weight , Receptor for Advanced Glycation End Products , Receptors, Immunologic/biosynthesis , Receptors, Immunologic/chemistry , Recombinant Proteins/biosynthesis , Regression Analysis , Ribose/chemistry , Ribose/metabolism , Serum Albumin, Bovine/chemistry , Serum Albumin, Bovine/metabolism , Spectrometry, FluorescenceABSTRACT
Dipeptidyl peptidase IV (DPP-IV) inhibition has the potential to become a valuable therapy for type 2 diabetes. We report the first use of solid-phase synthesis in the discovery of a new DPP-IV inhibitor class and a solution-phase synthesis that is practical up to the multikilogram scale. One compound, NVP-DPP728 (2), is profiled as a potent, selective, and short-acting DPP-IV inhibitor that has excellent oral bioavailability and potent antihyperglycemic activity.
