The nonenzymatic glycation of proteins is a ubiquitous process related to pathophysiology in diabetes mellitus and normal aging.  The most studied of the non-enzymatic glycation reactions involves glucose binding with human hemoglobin, HbA.  Five interconverting glucose structures generated upon mutarotation within the erythrocyte undergo transient, non-covalent and reversible binding to HbA.  This first stage of a four-stage process is followed by the formation of Schiff base, Amadori intermediate, and an array of glycation end products ultimately yielding structurally-modified proteins.  The overall rate-determining step precedes Amadori formation, resides within the early non-covalent stages, and is unknown. Computational modeling and nuclear magnetic resonance spectroscopy were used to assess pre-Amadori mechanistic possibilities with potential rate-determining implications.  Intracellular facilitating reagents inorganic phosphate, bicarbonate, lactate, and water can transiently and concomitantly bind in the same HbA cavity as the initially-bound cyclic glucose anomer.  These facilitating reagents can also enhance bound glucose ring opening to the transient ringed-opened isomer of glucose promoting the formation of bound Schiff base.  Multiple mechanistic roles for the facilitating reagents exist because the identity and the number of facilitating reagents and the geometry between the concomitantly bound reagents relative to each other and to proximate amino acid residues varies from protein cavity to protein cavity and can differ based upon the identity of the initially-bound anomer (a- versus �-pyranose).  The parameters that may dictate site specificity and the overall rate of HbA glycation in the non-covalent/pre-Amadori stages are a) the relative and absolute concentrations of phosphate, bicarbonate, and lactate, b) cavity-specific water availability, and c) the partitioning between available a- versus �-pyranose.

Multiple mechanisms in the initial noncovalent binding of glucose to human hemoglobin in nonenzymatic glycation

248th National Meeting (2014)

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