New evidence for the diversity of mechanisms and protonated Schiff bases formed in the non-enzymatic covalent protein modification (NECPM) of HbA by the hydrate and aldehydic forms of acetaldehyde and glyceraldehyde

Figures & data

Figure 1. Structures of salient species in the assessment of the nonenzymatic covalent protein modification of HbA by acetaldehyde or glyceraldehyde, with potential effector reagents 2,3-bisphosphoglycerate (2,3-BPG) and inorganic phosphate (Pi).

Structures 1 and 2 represent the aldehydic and hydrate forms of acetaldehyde. The relative length of the equilibrium arrows indicate the relative equilibrium concentrations of each form in aqueous solution (33% aldehyde, 66% hydrate). Structures 3 and 4 represent the aldehydic and hydrate forms of glyceraldehyde that exist in equilibrium (4% aldehyde, 96% hydrate). Structures 5 and 6 represent di-anionic and tetra-anionic 2,3-bisphosphoglycerate (2,3-BPG) that exist in equilibrium based upon pH. Structures 7 and 8 represent two forms of inorganic phosphate: mono-anionic Pi, 7, and di-anionic Pi, 8, both of which exist under physiological conditions.

Figure 2. Generic scheme for the nonenzymatic covalent protein modification (NECPM) of a protein by a non-glucose binding species.

The semi-circle represents a generic protein pocket in which a non-glucose species binds. Here the scheme represents a protein pocket that includes the N-terminus. The NECPM process can proceed in internal protein pockets as well. Once bound in the pocket, the binding species, serving as an electrophile, can undergo nucleophilic attack by a lone pair on an amine group of an amino acid residue (shown here as an N-terminus amino acid) which ultimately results in covalent attachment to the protein via variable mechanisms.

Figure 3. Potential NECPM mechanisms for covalent modification of Val1 of HbA that are geometrically possible for acetaldehyde 1 (Mechanism I) and the acetaldehyde hydrate 2 (Mechanisms II and III) based upon molecular modeling with MOE.¹

These mechanisms were predicted to be geometrically able to proceed based upon energetic minima calculated from MOE. In the top box, mechanism I, species 1 binds and then the nucleophilic lone pair on the nitrogen of a terminal valine amino acid residue attacks the carbonyl carbon of 1. This causes the π-bond of the carbonyl to break, enhancing the basicity of the carbonyl oxygen, which then abstracts a proton from a nearby acidic amino acid residue (Lys NH₃⁺ or a protonated histidine). This forms the protonated carbinol amine. A basic amino acid residue can then deprotonate the amine, generating the carbinol amine. The lone pair on the nitrogen can then form a π-bond, as the alcohol group (hydoxyl group) abstracts a proton from a nearby acidic amino acid residue and departs as water. This forms the protonated Schiff base, 9. Mechanisms II and III begin with the hydrate form of acetaldehyde, 2, bound in the protein pocket. In Mechanism II, the oxygen of an alcohol group on 2 abstracts a proton from a nearby acidic amino acid residue. The lone pair on the nitrogen of a terminal valine then attacks as a nucleophile, causing water to depart as a leaving group. This generates the protonated carbinol amine. In Mechanism III, the oxygen of an alcohol group on 2 abstracts a proton from a nearby acidic amino acid residue. The lone pair on the oxygen of the remaining alcohol group of 2 forms a π-bond to carbon. This facilitates the loss of water as a leaving group and generates an activated carbonyl species. The carbonyl carbon is then attacked by a nucleophilic lone pair on the nitrogen of a terminal valine to form the protonated carbinol amine. Both Mechanisms II and III involve an identical protonated carbinol amine that leads to the formation of the same protonated Schiff base, 9.

Table 1. Computational modeling of acetaldehyde (aldehyde 1 and hydrate 2) binding in the HbA_1c pocket of human hemoglobin as both single-species binding and concomitant binding with potential effector reagents, 2,3-BPG (5 or 6) or Pi (7 or 8)

Bound Species	Second Species Bound	Environment based upon charge state of Amino Acid Residues	Binding Energies (kcal/mol)	% of Binding Events with Geometry Suitable for Reaction to Modify Val1	Identity of the Acid involved in Facilitating Covalent Modification
Acetaldehyde 1	–	1	−3.2 to −2.7	3.3%	Lys82
	–	2	–3.2 to −2.7	26.6%	Lys8 or Lys82 or His2
	–	3	–3.4 to −2.8	10.0%	Lys8 or Lys82
	–	4	–3.2 to −2.7	23.3%	Lys8 or Lys82 or His2
	–	5	–3.0 to −2.5	13.3%	Lys8 or Lys82
		AVG		15.3%
Acetaldehyde Hydrate 2	–	1	−3.4 to −2.8	16.6%	Lys82 or Lys8
	–	2	–3.5 to −2.9	6.6%	Lys82 or Lys8 or His2
	–	3	–3.6 to −2.8	6.6%	Lys82 or Lys8
	–	4	–3.7 to −3.0	6.6%	Lys82 or His2
	–	5	–3.4 to −2.9	0.0%	N/A
		AVG		7.3%
Acetaldehyde 1	2,3-BPG^2-	1	−3.0 to −2.6	7.4%	Lys8 or 2,3-BPG^2-
	2,3-BPG^4-	1	–3.3 to −2.7	6.7%	Lys82 or 2,3-BPG^4-*
	Pi^−	1	–3.2 to −2.7	0.0%	N/A
	Pi^2-	1	–3.0 to −2.7	0.0%	N/A
		AVG		3.5%
Acetaldehyde Hydrate 2	2,3-BPG^2-	1	−3.5 to −2.8	26.6%	2,3-BPG^2- or Lys82 or Lys8
	2,3-BPG^4-	1	–3.8 to −2.9	16.6%	Lys8 or 2,3-BPG^4-* or Lys82
	Pi^−	1	–3.5 to −2.8	6.6%	Lys82 or Lys8
	Pi^2-	1	–3.5 to −2.9	6.6%	Lys82
		AVG		14.2%

Table 1 contains data from the computational modeling of acetaldehyde (1) and its hydrate (2) binding in the HbA_1c pocket of human hemoglobin, both as single-species binding and with concomitant binding with either form of 2,3-BPG (5 or 6) or Pi (7 or 8). Column 1 describes which form of acetaldehyde (1 or 2) participates in binding. Column 2 describes which species (if any) was concomitantly bound in the pocket. Column 3 describes the environment of the HbA_1c pocket in which binding took place (charge states are defined at the end of Table 1 Notes). Column 4 describes the range of exothermicity (in kcal/mol) with which the substrate bound in the pocket. Note that for concomitant binding, the overall exothermicity is the sum of the two binding events. As an example, for 1 binding with 2,3-BPG^2- total exothermicity is (−3.2 to −2.7) + (−3.0 to −2.6) or ca. −6.2 to −5.3 kcal/mol. Column 5 describes the percentage of binding events with suitable geometry for nucleophilic attack by a Val1 residue (relative to the total number of binding events that were energetic minima). Column 6 describes the identity of the acid that facilitates the Val1 attack. Note that in column 6, the acids are listed in the order of decreasing % of binding events that meet the geometric requirements for participation. *Indicates that the species is acting as a proton shuttle (abstracting a proton off of an acidic residue and then acting as an acid).

Environment 1—Standard Environment: Val(NH₂), Lys(NH₃⁺), Glu/Asp(COO⁻), His(Neutral; 146-H⁺).

Environment 2—H-Bond Donating/Acidic Environment: Val(NH₂), Lys(NH₃⁺), Glu/Asp(COOH), His(H⁺).

Environment 3—H-Bond Accepting/Basic Environment: Val(NH₂), Lys(NH₃⁺), Glu/Asp(COO⁻), His(Neutral).

Environment 4—Mixed Environment 1: Val(NH₂), Lys(NH₃⁺), Glu/Asp(COO⁻), His(H⁺).

Environment 5—Mixed Environment 2: Val(NH₂), Lys(NH₃⁺), Glu/Asp(COOH), His(Neutral).

Table 2. Computational modeling of Glyceraldehyde/Hydrate binding to the HbA_1c pocket of human hemoglobin both as single-species binding and with concomitant binding with 2,3-BPG or with Pi

Bound Species	Second Species Bound	Environment based upon charge state of Amino Acid Residues	Binding Energies (kcal/mol)	% of Binding Events with Geometry Suitable for Reaction to Modify Val1	Identity of the Acid involved in Facilitating Covalent Modification
Glyceraldehyde 3	–	1	−3.6 to −3.1	46.7%	Lys82 or Lys8
	–	2	−3.5 to −3.3	13.3%	Lys8 or Lys82
	–	3	−3.0 to −2.9	3.3%	Lys8
	–	4	−3.7 to −3.4	26.6%	Lys8 or Lys82
	–	5	−3.6 to −3.2	0.0%	N/A
		AVG		18.0%
Glyceraldehyde Hydrate 4	–	1	−3.6 to −3.1	30.0%	Lys82
	–	2	−3.8 to −3.2	0.0%	N/A
	–	3	−3.8 to −3.0	0.0%	N/A
	–	4	−4.0 to −3.3	33.3%	Lys8 or Lys82
	–	5	−3.7 to −3.3	0.0%	N/A
		AVG		12.7%
Glyceraldehyde 3	2,3-BPG^2-	1	−3.8 to −2.8	26.7%	Lys8 or 2,3-BPG^2- or Lys82
	2,3-BPG^4-	1	−4.1 to −3.1	23.3%	Lys82 or 2,3-BPG^4-*
	Pi^−	1	−3.8 to −2.8	20.0%	Lys8 or Lys82
	Pi^2-	1	−3.7 to −3.0	0.0%	N/A
		AVG		17.5%
Glyceraldehyde Hydrate 4	2,3-BPG^2-	1	−3.8 to −2.9	33.3%	Lys82 or 2,3-BPG^2- or Lys8
	2,3-BPG^4-	1	−4.2 to −3.2	10.0%	Lys82
	Pi^−	1	−3.9 to −2.8	13.3%	Lys82 or Lys 8
	Pi^2-	1	−3.9 to −2.8	0.0%	N/A
		AVG		14.1%

Table 2 contains data from the computational modeling of glyceraldehyde (3) and its hydrate (4) binding to the HbA_1c pocket of human hemoglobin, both as single-species binding and with concomitant binding with either form of 2,3-BPG (5 or 6) or Pi (7 or 8). Column 1 describes which form of glyceraldehyde (3 or 4) participates in binding. Column 2 describes which species (if any) was concomitantly bound in the pocket. Column 3 describes the environment of the HbA_1c pocket in which binding took place (charge states are defined at the end of Table 1 Notes). Column 4 describes the range of exothermicity (in kcal/mol) with which the substrate bound in the pocket. Note that for concomitant binding, the overall exothermicity is the sum of the two binding events. As an example, for 3 binding with 2,3-BPG^2- total exothermicity is (−3.6 to −3.1) + (−3.8 to −2.8) or ca. −7.4 to −5.9 kcal/mol. Column 5 describes the percentage of binding events with suitable geometry for nucleophilic attack by a Val1 residue (relative to the total number of binding events that were energetic minima). Column 6 describes the identity of the acid that facilitates the Val1 nucleophilic attack. Note that in column 6, the acids are listed in the order of decreasing % of binding events that meet the geometric requirements for participation.

*Indicates that the species is acting as a proton shuttle (abstracting a proton off of an acidic residue and then acting as an acid).

Environment 1—Standard Environment: Val(NH₂), Lys(NH₃⁺), Glu/Asp(COO⁻), His(Neutral; 146-H⁺).

Environment 2—H-Bond Donating/Acidic Environment: Val(NH₂), Lys(NH₃⁺), Glu/Asp(COOH), His(H⁺).

Environment 3—H-Bond Accepting/Basic Environment: Val(NH₂), Lys(NH₃⁺), Glu/Asp(COO⁻), His(Neutral).

Environment 4—Mixed Environment 1: Val(NH₂), Lys(NH₃⁺), Glu/Asp(COO⁻), His(H⁺).

Environment 5—Mixed Environment 2: Val(NH₂), Lys(NH₃⁺), Glu/Asp(COOH), His(Neutral).

Figure 4. Potential NECPM mechanisms for HbA covalent modification that are geometrically possible for glyceraldehyde 3 (Mechanism I) and the glyceraldehyde hydrate 4 (Mechanisms II and III) based upon molecular modeling with MOE.²

These three mechanisms are predicted to be geometrically feasible based upon calculated energetic minima from computations conducted in MOE. In Mechanism I, the aldehydic form of glyceraldehyde, 3, binds and the nucleophilic lone pair on the nitrogen of a terminal valine amino acid residue attacks the carbonyl carbon of 3. This causes the π-bond to break and enhances the basicity of the carbonyl oxygen, which then abstracts a proton from a nearby acidic amino acid residue (Lys NH₃⁺ or a protonated histidine). This generates the protonated carbinol amine. Once formed, a basic amino acid residue can deprotonate the positively charged amine to form the carbinol amine. The lone pair on nitrogen can then form a π-bond, increasing the basicity of the oxygen of the alcohol which can then abstract a proton from an acidic amino acid reside. The protonated alcohol group then departs as water. This forms the protonated Schiff base, 10. Mechanisms II and III begin with the hydrate form of glyceraldehyde, 4, bound in the protein pocket. In Mechanism II, the oxygen of an alcohol group on 4 abstracts a proton from a nearby acidic amino acid residue. The lone pair on the nitrogen of a terminal valine then attacks as a nucleophile, causing water to depart as a leaving group. This generates the protonated carbinol amine. In Mechanism III, the oxygen of an alcohol group on 4 abstracts a proton from a nearby acidic amino acid residue. The lone pair on the oxygen of the remaining alcohol group of 4 forms a π-bond to carbon. This facilitates the loss of water as a leaving group and generates an activated carbonyl species. The carbonyl carbon is then attacked by a nucleophilic lone pair on the nitrogen of a terminal valine to form the protonated carbinol amine. Both Mechanisms II and III involve a common protonated carbinol amine species and the following chemistry from the common intermediate leads to the formation of the same protonated Schiff base, 10.