Raja G. Khalifah

In Vitro Kinetic Studies of Formation of Antigenic Advanced Glycation End Products (AGEs) NOVEL INHIBITION OF POST-AMADORI GLYCATION PATHWAYS

Nonenzymatic protein glycation (Maillard reaction) leads to heterogeneous, toxic, and antigenic advanced glycation end products (“AGEs”) and reactive precursors that have been implicated in the pathogenesis of diabetes, Alzheimers disease, and normal aging. In vitro inhibition studies of AGE formation in the presence of high sugar concentrations are difficult to interpret, since AGE-forming intermediates may oxidatively arise from free sugar or from Schiff base condensation products with protein amino groups, rather than from just their classical Amadori rearrangement products. We recently succeeded in isolating an Amadori intermediate in the reaction of ribonuclease A (RNase) with ribose (Khalifah, R. G., Todd, P., Booth, A. A., Yang, S. X., Mott, J. D., and Hudson, B. G. (1996) Biochemistry 35, 4645-4654) for rapid studies of post-Amadori AGE formation in absence of free sugar or reversibly formed Schiff base precursors to Amadori products. This provides a new strategy for a better understanding of the mechanism of AGE inhibition by established inhibitors, such as aminoguanidine, and for searching for novel inhibitors specifically acting on post-Amadori pathways of AGE formation. Aminoguanidine shows little inhibition of post-Amadori AGE formation in RNase and bovine serum albumin, in contrast to its apparently effective inhibition of initial (although not late) stages of glycation in the presence of high concentrations of sugar. Of several derivatives of vitamins B1 and B6 recently studied for possible AGE inhibition in the presence of glucose (Booth, A. A., Khalifah, R. G., and Hudson, B. G. (1996) Biochem. Biophys. Res. Commun. 220, 113-119), pyridoxamine and, to a lesser extent, thiamine pyrophosphate proved to be novel and effective post-Amadori inhibitors that decrease the final levels of AGEs formed. Our mechanism-based approach to the study of AGE inhibition appears promising for the design and discovery of novel post-Amadori AGE inhibitors of therapeutic potential that may complement others, such as aminoguanidine, known to either prevent initial sugar attachment or to scavenge highly reactive dicarbonyl intermediates.

Unusual susceptibility of heme proteins to damage by glucose during non-enzymatic glycation

Glucose modifies the amino groups of proteins by a process of non-enzymatic glycation, leading to potentially deleterious effects on structure and function that have been implicated in the pathogenesis of diabetic complications. These changes are extremely complex and occur very slowly. We demonstrate here that hemoglobin and myoglobin are extremely susceptible to damage by glucose in vitro through a process that leads to complete destruction of the essential heme group. This process appears in addition to the expected formation of so-called advanced glycation end products (AGEs) on lysine and other side-chains. AGE formation is enhanced by the iron released. In contrast, the heme group is not destroyed during glycation of cytochrome c, where the sixth coordination position of the heme iron is not accessible to solvent ligands. Glycation leads to reduction of ferricytochrome c in this case. Since hydrogen peroxide is known to destroy heme, and the destruction observed during glycation of hemoglobin and myoglobin is sensitive to catalase, we propose that the degradation process is initiated by hydrogen peroxide formation. Damage may then occur through reaction with superoxide generated (a reductant of ferricytochrome c), or hydroxyl radicals, or with both.

Post-Amadori AGE inhibition as a therapeutic target for diabetic complications: a rational approach to second-generation Amadorin design.

Abstract: Aminoguanidine and pyridoxamine (Pyridorin™), two major inhibitors of advanced glycation end product (AGE) formation, have entered clinical trials for diabetic nephropathy. They share no structural similarity and are believed to inhibit AGE formation by entirely different mechanisms. Pyridoxamine is a post‐Amadori AGE inhibitor—that is, an “Amadorin”—whereas aminoguanidine primarily scavenges reactive dicarbonyl precursors to AGEs. However, pyridoxamine also has a limited potential to react with dicarbonyls. We thus embarked on an effort to develop second‐generation Amadorins with low nucleophilicity. Our hypothesis was that we could improve specificity for inhibiting the post‐Amadori pathway by minimizing the potential for scavenging small dicarbonyl intermediates. This mechanism‐based strategy has led to a rational drug design program that has successfully produced candidate Amadorins, among them the novel compound BST‐4997. This Amadorin has greater post‐Amadori potency than pyridoxamine but possess no dicarbonyl scavenging activity. Prototypical inhibitors like BST‐4997 provide a unique tool to help identify relevant AGE pathways that contribute to diabetic complications. Targeting AGE inhibition differs significantly from traditional approaches to drug discovery and thus represents a new paradigm for the drug industry that should be recognized.

13C-NMR and spectrophotometric studies of alcohol-lipid interactions.

The interactions of butanol and mixtures of butanol and ethanol with dipalmitoylphosphatidyl choline (DPPC) liposomes have been investigated by both spectrophotometric measurements and Fourier transform 13C nuclear magnetic resonance spectroscopy. The spectrophotometric experiments indicate that butanol exhibits the same effects on the thermotropic properties of DPPC as the other short chain alcohols, methanol, ethanol and propanol, which have been shown to be characteristic of the alcohol induced transition of the lipid to the interdigitated state. An additive effect of butanol and ethanol on the induction of the interdigitated phase in DPPC was also observed. A decrease in line width and increase in T1 of the choline methyl signal were observed in the 13C-NMR experiments conducted at 32 degrees C when butanol was added to DPPC in increasing amounts suggesting an increase of disorder in the head group region of the lipid. Addition of ethanol to the NMR sample containing butanol produced hysteresis in the heating and cooling curves characteristic of the interdigitated state. In the interdigitated state, the choline methyl signal exhibited a T1 value equal to that when the lipid is in the fluid state. The increase of mobility in the head group region in the interdigitated gel state relative to the bilayer gel can be rationalized by the increase in surface area in that site when the lipid interdigitates.

Reflections on Edsall's carbonic anhydrase: paradoxes of an ultra fast enzyme

John Edsalls investigations of human erythrocyte carbonic anhydrase, a zinc metalloenzyme that powerfully catalyzes the reversible hydration of carbon dioxide, highlighted a conundrum regarding the correct hydration product. The measured kinetic parameters could not be reconciled with the choice of carbonic acid, since its bimolecular recombination rate with enzyme would exceed the diffusion limit. The alternate choice of bicarbonate obviated the recombination rate problem but required that the active site deprotonation exceed the diffusion-limited maximum rate by an even greater extent. This paradox was resolved in favor of bicarbonate when the unsuspected role of buffer species indirectly deprotonating the enzyme was finally proposed, spurring numerous investigations to verify the hypothesis. Edsalls laboratory also reported the accidental discovery of the first competitive inhibitor, imidazole. This opened new avenues to understanding the binding of the CO(2) substrate and stimulated many investigations on this inhibitor. Paramagnetic NMR and crystallographic studies demonstrated that the only other known competitive inhibitor, phenol, apparently shared this unusual binding site. Despite enormous progress since Edsalls retirement, particularly the use of site-directed mutagenesis approaches, the precise interactions of carbon dioxide and bicarbonate with specific active site moieties remain as elusive today as when Edsall first considered these questions.

Molecular Basis for Catalytic Activity Changes in Active‐Site‐Modified Carbonic Anhydrases: A13C Magnetic Resonance View

The understanding of the catalytic mechanism of enzyme action requires the synthesis of diverse types of information, such as kinetic characterization, crystal structure determination, and the effects of metal-ion replacement, coenzyme analogue substitutions, and chemical modification of various active-site residues. In addition, chemical modification for amino acid sequence determination is a prerequisite for many of these types of studies. It is thus inconceivable that an understanding of the mechanism of action of any enzyme can be achieved without extensive modification of its structure. Nevertheless, there is often considerable apprehension about the study of the catalytic activity of active-site modified enzymes and about the relevance of such studies to the understanding of the unmodified forms. The basis for such caution is usually the extreme sensitivity of catalytic activity and kinetics to very small structural perturbations or charge alterations. Consequently, the interpretation of observed changes in catalytic activity and kinetics in terms of specific alterations in the active-site structure requires much supporting evidence. Such endeavors are quite essential, however, since not all proposed mechanisms for the unmodified enzymes can rationalize the observed consequences of the active-site modifications. The study of covalently and specifically modified enzyme forms can thus be regarded as an attempt to produce by design a set of pseudoisozymes whose comparative study may lead to useful structure-function correlations similar to those often sought in the comparative study of naturally occumng isozymes or structurally homologous enzymes that show distinct kinetic differences.