Potential roles of inorganic phosphate on the progression of initially bound glucopyranose toward the nonenzymatic glycation of human hemoglobin: Mechanistic diversity and impacts on site selectivity

Abstract

Nonenzymatic glycation (NEG) begins with the non-covalent binding of a glucopyranose to a protein. The bound glucopyranose must then undergo structural modification to generate a bound electrophile that can reversibly form a Schiff base, which can then lead to Amadori intermediates, and ultimately to glycated proteins. Inorganic phosphate (Pi) is known to accelerate the glycation of human hemoglobin (HbA), although the specific mechanism(s) of Pi as an effector reagent have not been determined. The aim of this study was to determine whether Pi and a glucopyranose can concomitantly bind to HbA and react while bound within the early, noncovalent stages to generate electrophilic species capable of progress in NEG. 31P and 1HNMR of model reactions confirm that bimolecular reactions between Pi and glucopyranose occur generating modified glucose electrophiles. Computations of protein/substrate interactions predict that Pi can concomitantly bind with a glucopyranose in HbA pockets with geometries suitable for multiple acid/base mechanisms that can generate any of four transient electrophiles. Pi-facilitated mechanisms in the noncovalent stages predict that the glycation of β-Val1 of HbA to HbA1c is a “hot spot” because the β-Val1 pocket facilitates many more mechanisms than any other site. The mechanistic diversity of the Pi effect within the early noncovalent stages of NEG predicts well the overall site selectivity observed from the in vivo glycation of HbA in the presence of Pi. These insights extend our basic understanding of the NEG process and may have clinical implications for diabetes mellitus and even normal aging.

Does Protein Glycation Impact on the Drought-Related Changes in Metabolism and Nutritional Properties of Mature Pea (Pisum sativum L.) Seeds?

“…Reducing sugars, aldoses and ketoses, readily react with lysyl residues of proteins forming Schiff base adducts, which readily undergo further Amadori or Heyns rearrangements to yield keto-and aldoamines, respectively [2,3] ( Figure 1). Carbohydrate derivatives-sugar phosphates [4,5], sugar acids [6] and nucleotides [7]-were also reported as glycation agents. The products of early glycation, also known as Amadori and Heyns compounds, can be involved in oxidative degradation, often referred to as glycoxidation [8], yielding a structurally diverse group of advanced glycation end products (AGEs) [9].…”

Insights into the Progression of Labile Hb A1cto Stable Hb A1cviaa Mechanistic Assessment of 2,3-Bisphosphoglycerate Facilitation of the Slow Nonenzymatic Glycation Process

“…This methodology may be explained by the Le Chatelier and Braun principle [30], which states that if a reactant's concentration is decreased in a dynamic reversible equilibrium, the equilibrium will shift to the left (towards starting materials). Our computational data predicts that as phosphate is added, it binds HbA in competition with a glucopyranose [26]. In some clinical assays of stable Hb A 1c , phosphate has been added to hemolyzing solutions at levels well above physiological values (e.g.…”

“…the amine form of the Nterminal Val1 on the two β chains of Hb A) reacts with the electrophilic ring-opened form of glucose (structure 2). Moreover, several studies have shown that the rate of structure 3 formation is influenced by effector reagents such as inorganic phosphate and water serving as acids and/or bases [3,20,25,26].…”

“…Thus, stable Hb A 1c formation in the absence of 2,3-BPG and any other effector reagent should be extremely slow. However, it should be noted from earlier studies that physiological inorganic phosphate (existing at concentrations of 0.5-0.8 mM in the erythrocyte) [27] can bind with glucose in the Hb A 1c pocket and serve as an effector reagent, increasing the rate of Hb A glycation [26]. Moreover, it is estimated that 67.0% of all Hb A 1c pockets in erythrocytes possess a bound 2,3-BPG [28].…”

“…In the noncovalent stages of NEG, a glucopyranose molecule structure 1, must first enter the Hb A 1c protein pocket and then be modified to ring-opened species structure 2 while in the pocket, so it can become sufficiently reactive [4]. Structure 2 can then undergo multiple reactions to covalently bond to the N-terminal valine and become structure 3, the conjugate acid of the Schiff base which is labile Hb A 1c [26]. Mechanistic pathway I: in structure 3 (labile Hb A 1c ), the asterisk (*) denotes the C2 atom.…”