Cyclopropylglyoxylate as a mechanistic probe of thiamine pyrophosphate dependent pyruvate-metabolizing enzymes

Four pyruvate-decarboxylating enzymes with thiamine pyrophosphate (TPP) cofactors catalyze the decarboxylation of the cyclopropyl substrate analog cyclopropylglyoxylate. Pyruvate: ferredoxin oxidoreductase, an archaebacterial enzyme which catalyzes oxidation of the hydroxyethyl-TPP (HETPP) intermediate by two one-electron transfers to an iron-sulfur center, generates the coenzyme A thioester of cyclopropylcarboxylic acid. A long-lived free radical, HETPP is thought to be an intermediate in the pyruvate to acetyl-CoA conversion; however, cleavage of the cyclopropyl ring was not detected. Pyruvate decarboxylase, pyruvate oxidase, and pyruvate dehydrogenase also generate the corresponding cyclopropyl products. The applicability of cyclopropyl substrate analogs as indicators of free-radical enzyme mechanisms is discussed in light of these results.     

2-Keto-3-fluoroglutarate: a useful mechanistic probe of 2-keto-glutarate-dependent enzyme systems

2-Keto-3-fluoroglutaric acid prepared by acid hydrolysis of its diethyl ester is stable, as the free acid in aqueous solution at pH 2, and can be stored at -20 degrees C for several years. Both enantiomers are reduced by NADH in the presence of glutamate dehydrogenase (EC 1.4.1.2) to the two diastereomers of 3-fluoro-L-glutamate, which are stable at neutral pH and at high pH unless heated. 2-Keto-3-fluoroglutarate exists in solution almost entirely as a hydrate both at low and neutral pH. Both enantiomers of ketofluoroglutarate react with the pyridoxamine forms of aspartate, alanine and 4-aminobutyrate transaminases to give fluoride release. 2 mol of cosubstrate amino acid react for each mol of ketofluoroglutarate (KFG) when starting from the pyridoxamine form of the enzyme: 2 RCHNH2COOH + KFG + H2O----F- + NH4+ + glutamate + 2 RCOCOOH. Both diastereomers of fluoroglutamate are decarboxylated by glutamate decarboxylase (EC 4.1.1.15) with fluoride release: KFG + H2O----CO2 + F- + HCOCH2CH2COOH. By contrast, only one isomer of fluoroglutamate will react with the pyridoxal form of glutamate-oxalacetate transaminase to give fluoride release: HOOCCHNH2CHFCH2COOH + H2O----4F- + NH4+ + HOOCCOCH2CH2COOH. The enzymatic decarboxylation of 3-fluoroisocitrate produces only one enantiomer of ketofluoroglutarate, which is reduced to threo (2R,3R)-3-fluoroglutamate by NADH and glutamate dehydrogenase: [2R,3S]-HOOCCH(OH)CF(COOH)CH2COOH + NADP+----[3R]-KFG + CO2 + NADPH + H+. The proton, 13C, and 19F-NMR parameters of ketofluoroglutarate and the two fluoroglutamate diastereomers are presented. These molecules are useful probes of enzymatic mechanisms thought to involve carbanion intermediates.     

Gaulin homogenization: a mechanistic study

Free radical-based oxidation has been detected in the normal operating regime of the Gaulin homogenizer, demonstrating that cavitation occurs in this important industrial bioprocessing equipment. Free radical generation is suppressed by imposition of back pressure, proving that such cavitation occurs in the impingement section. The calculated value of the cavitation number is consistent with submerged jet cavitation, wherein a high-speed jet exiting from the valve gap accelerates fluid in the impingement region, creating the vacuum conditions for cavitation. Using polysaccharides as a model shear-sensitive compound, their breakage pattern in the homogenizer was characterized by molecular size and polydispersity and compared to those of fluid shear flows in capillary tubes and cavitating flow from a sonic horn. The results indicate that breakage occurs primarily by fluid shear, although a contribution by cavitation is also apparent when back pressure is applied. Because biological molecules can readily react with free radicals and the alterations caused thereby are subtle in nature, a thorough evaluation of the impact of free radicals in upstream homogenization is warranted.     

Formycin triphosphate-terbium complex: a novel spectroscopic probe for phosphoryl transfer enzymes

The conditions under which the fluorescent pyrazolopyrimidine nucleotide formycin A triphosphate (7-amino-3-(beta-D-(5'- tripolyphosphate)ribofuranosyl)pyrazolo[4,3-d]pyrimidine, FTP) forms a 1:1 complex in solution with Tb3+ have been characterized. The complex has a dissociation constant of approx. 10(-7) M. Within the complex, the luminescence of Tb3+ is dramatically sensitized by energy transfer from formycin. The value for 50% transfer efficiency, F?rster's R0 (F?rster, T. (1964) in Modern Quantum Chemistry (Sinanoglu, O., ed.), pp. 93-137, Academic Press, New York) was determined to be 3.34 +/- 0.4 A, and the effective distance between the donor and acceptor transition dipoles, R, in the complex was estimated to be 6.6 +/- 1.0 A. The quantum yield of Tb3+ in the complex is sensitive to the number of O-H oscillators bound to the Tb3+, which allows determination of the number of waters bound to it (approx. 4). Preliminary results show that the complex binds to the phosphoryl transfer enzyme hexokinase in the presence of the glucose analogs N-acetylglucosamine, frucose and xylose, which are not phosphorylated by the enzyme. The binding occurs with a loss of energy efficiency consistent with a new distance from the effective transition dipole of formycin to that of terbium of approx. 9.6 A. The FTP-terbium complex can be used as both a spectroscopic and an X-ray diffraction probe. Studies with this compound should be most valuable for correlating solution and crystallographic data.     

Catalysis by human leukocyte elastase: proton inventory as a mechanistic probe

Proton inventories (rate measurements in mixtures of H2O and D2O) were determined for the human leukocyte elastase catalyzed hydrolyses of thiobenzyl esters and p-nitroanilides of the peptides MeOSuc-Val, MeOSuc-Alan-Pro-Val (n = 0-2), and MeOSuc-Alan-Pro-Ala (n = 1 or 2). The dependencies of k2/Ks on mole fraction of solvent deuterium for the p-nitroanilides are "dome-shaped" and were fit to a model that incorporates the mechanistic features of generalized solvent reorganization when substrate binds to enzyme and partial rate limitation of k2/Ks by physical and chemical steps [Stein, R. L. (1985) J. Am. Chem. Soc. 107, 7768-7769]. The proton inventories for the deacylation of MeOSuc-Val-HLE and MeOSuc-Pro-Val-HLE are linear while those for the deacylation of MeOSuc-Ala-Pro-Val-HLE and MeOSuc-Ala-Ala-Pro-Val-HLE are "bowl-shaped" and could be fit to a quadratic dependence of rate on mole fraction of deuterium. These results are interpreted to suggest that the correct operation of the catalytic triad is dependent on substrate structure. Minimal substrates, which cannot interact with elastase at remote subsites, are hydrolyzed via a mechanism involving simple general-base catalysis by the active site histidine and transfer of a single proton in the rate-limiting transition state. In contrast, tri- and tetrapeptide substrates, which are able to interact at remote subsites, are hydrolyzed by a more complex mechanism of protolytic catalysis involving full functioning of the catalytic triad and transfer of two protons in the rate-limiting transition state. Finally, the proton inventories for the deacylation of MeOSuc-Ala-Pro-Ala-HLE and MeOSuc-Ala-Ala-Pro-Ala-HLE are dome-shaped and suggest that the chemical events of acyl-enzyme hydrolysis are only partially rate limiting for these reactions and that some other physical step is also partially rate limiting.     

Post-translational self-hydroxylation: a probe for oxygen activation mechanisms in non-heme iron enzymes

Recent years have seen considerable evolution in our understanding of the mechanisms of oxygen activation by non-heme iron enzymes, with high-valent iron-oxo intermediates coming to the forefront as formidably potent oxidants. In the absence of substrate, the generation of vividly colored chromophores deriving from the self-hydroxylation of a nearby aromatic amino acid for a number of these enzymes has afforded an opportunity to discern the conditions under which O2 activation occurs to generate a high-valent iron intermediate, and has provided a basis for a rigorous mechanistic examination of the oxygenation process. Here, we summarize the current evidence for self-hydroxylation processes in both mononuclear non-heme iron enzymes and in mutant forms of ribonucleotide reductase, and place it within the context of our developing understanding of the oxidative transformations accomplished by non-heme iron centers.     

The enediolate analogue 5-phosphoarabinonate as a mechanistic probe for phosphoglucose isomerase.

A stable analogue has been prepared of the enediolate anion believed to occur transiently in the reaction of phosphoglucose isomerase. This compound, 5-phosphoarabinonate, is the strongest known competitive inhibitor of the enzyme (Ki = 3 times 10(-7) M below pH 7). A distinctive pH dependence of binding, also found for two other aldonic acid omega-phosphates, 6-phosphogluconate and 4-phosphoerythronate, involves pertubation of a pKa from 7.0 in the free enzyme to 9.0 in the enzyme-inhibitor complex. This perturbation may reflect a catalytically advantageous increase in basicity which occurs around the transition state of the normal enzymatic reaction.     

Function of pyridoxal 5'-phosphate in glycogen phosphorylase: a model study using 6-fluoro-5'-deoxypyridoxal- and 5'-deoxypyridoxal-reconstituted enzymes

A new vitamin B6 analogue, 6-fluoro-5'-deoxypyridoxal (6-FDPL), was synthesized and characterized. This analogue, as well as 6-fluoropyridoxal (6-FPAL), 6-fluoropyridoxal phosphate (6-FPLP), and 6-fluoropyridoxine, showed positive heteronuclear 1H-19F nuclear Overhauser effects between the 5'-protons and the 6-fluorine. Apophosphorylase reconstituted with 6-FDPL showed 1% of the activity of the native enzyme in the presence of phosphite. The kinetic pattern, apparent pH optimum of activity, and the activity-temperature dependency of the 6-FDPL-enzyme were virtually identical with those of phosphorylase reconstituted with the parent compound, 6-FPAL [Chang, Y. C., & Graves, D. J. (1985) J. Biol. Chem. 260, 2709-2714], except the Km of phosphite toward the 6-FDPL-enzyme was 9 times higher than that with the 6-FPAL-enzyme and the 6-FDPL-enzyme showed a lower Vmax value. Phosphorylase reconstituted with 5'-deoxypyridoxal (DPL) also showed activity in the presence of phosphite. The kinetics and the temperature-activity dependency of this reconstituted enzyme were investigated. 19F nuclear magnetic resonance studies showed that the binding of glucose 1-phosphate to a 6-FDPL-enzyme-adenosine 5'-phosphate (AMP) complex shifted the 19F signal 0.6 ppm upfield, whereas a 2.1 ppm change was observed when the 6-FPAL-enzyme-AMP formed a complex with glucose 1-phosphate [Chang, Y. C., Scott, R. D., & Graves, D. J. (1986) Biochemistry 25, 1932-1939].(ABSTRACT TRUNCATED AT 250 WORDS)     

Hexacyanochromate ion as a paramagnetic anion probe for active sites of enzymes

Hexacyanochromate ion, (Cr(CN)6)3-, was applied to ribonuclease T1 (RNase T1), which specifically cleaves RNA chains at guanylic acid residues. From kinetic studies, this anion was shown to bind to the active site of RNase T1 as a competitive inhibitor. Therefore, the line broadening effect of NMR resonances due to binding of (Cr(CN)6)3- was analyzed for the mapping of the active site of RNase T1. His-40 C2 proton resonance was significantly broadened, following His-92 C2 proton resonance upon binding of (Cr(CN)6)3-, while His-27 C2 proton resonance did not show any appreciable line broadening. Moreover, from the pH dependence of the line broadening effect, the binding of (Cr(CN)6)3- was shown to be controlled by the ionic state of Glu-58. Based on the present NMR results and x-ray crystal structure, the active site structure of RNase T1 is discussed.     

Different mechanistic requirements for prokaryotic and eukaryotic chaperonins: a lattice study

MOTIVATION: The folding of many proteins in vivo and in vitro is assisted by molecular chaperones. A well-characterized molecular chaperone system is the chaperonin GroEL/GroES from Escherichia coli which has a homolog found in the eukaryotic cytosol called CCT. All chaperonins have a ring structure with a cavity in which the substrate protein folds. An interesting difference between prokaryotic and eukaryotic chaperonins is in the nature of the ATP-mediated conformational changes that their ring structures undergo during their reaction cycle. Prokaryotic chaperonins are known to exhibit a highly cooperative concerted change of their cavity surface while in eukaryotic chaperonins the change is sequential. Approximately 70% of proteins in eukaryotic cells are multi-domain whereas in prokaryotes single-domain proteins are more common. Thus, it was suggested that the different modes of action of prokaryotic and eukaryotic chaperonins can be explained by the need of eukaryotic chaperonins to facilitate folding of multi-domain proteins. RESULTS: Using a 2D square lattice model, we generated two large populations of single-domain and double-domain substrate proteins. Chaperonins were modeled as static structures with a cavity wall with which the substrate protein interacts. We simulated both concerted and sequential changes of the cavity surfaces and demonstrated that folding of single-domain proteins benefits from concerted but not sequential changes whereas double-domain proteins benefit also from sequential changes. Thus, our results support the suggestion that the different modes of allosteric switching of prokaryotic and eukaryotic chaperonin rings have functional implications as it enables eukaryotic chaperonins to better assist multi-domain protein folding.     

High density lipoprotein cholesterol as a mechanistic probe for the side chain cleavage reaction.

Cholesterol side chain cleavage reaction catalyzed by purified cytochrome P-450scc was stimulated 4-5 fold when cholesterol in rat high density lipoprotein was used as a substrate as compared to the case where cholesterol plus 0.1% Emulgen 911 was used. In the case of the cholesterol-Emulgen system, the Vmax value of activity was not obtained even when a 20 times molar excess of adrenodoxin over the cytochrome was used. However, in the case of the lipoprotein, a 2-3 times molar excess of adrenodoxin over the cytochrome was enough to obtain the half value of Vmax. HPLC gel filtration experiments showed that the three enzymes were eluted from the column as a complex regardless of adding the lipoprotein as judged by the activity and the blotting analysis. However, the activity with the lipoprotein was detected significantly earlier than that in the absence. These and other lines of evidence suggest that the lipoprotein vesicles promote a complex formation among the three enzyme components and serve as a probe for emphasizing a significance of a cluster mechanism in the reaction.     

Glutathione oxidase activity of selenocystamine: a mechanistic study.

Selenocystamine (RSe-SeR) was shown to catalyze the oxygen-mediated oxidation of excess GSH to glutathione disulfide, at neutral pH and ambient PO2. This glutathione oxidase activity required the heterolytic reduction of the diselenide bond, which produced two equivalents of the selenolate derivative selenocysteamine (RSe-), via the transient formation of a selenenylsulfide intermediate (RSe-SG). Formation of RSe- was the only reaction observed in anaerobic conditions. At ambient PO2, the kinetics and stoichiometry of GSSG production as well as that of GSH and oxygen consumptions demonstrated that RSe- performed a three-step reduction of oxygen to water. The first step was a one-electron transfer from RSe- to dioxygen, yielding superoxide and a putative selenyl radical RSe., which decayed very rapidly to RSe-SeR. In the second step, RSe- reduced superoxide to hydrogen peroxide through a much faster one-electron transfer, also associated with the decay of RSe. to RSe-SeR. The third step was a two-electron transfer from RSe- to hydrogen peroxide, again much faster than oxygen reduction, which resulted in the production of RSe-SG, presumably via a selenenic acid intermediate (RSeOH) which was trapped by excess GSH. This third step was studied on exogenous hydroperoxide in anaerobic conditions, and it could be eliminated from the glutathione oxidase cycle in the presence of excess catalase. The role of RSe- as a one- and two-electron reductant was confirmed by competitive carboxymethylation with iodoacetate. RSe- was able to rapidly reduce ferric cytochrome c to its ferrous derivative. The overall rate of catalytic glutathione oxidation was GSH concentration dependent and oxygen concentration independent. Excess glutathione reductase and NADPH increased the catalytic oxidation of GSH, probably by switching the rate-limiting step from selenylsulfide to diselenide cleavage. When GSH was substituted for dithiothreitol, it was shown to reduce RSe-SeR to RSe- in a fast and quantitative reaction, and selenocystamine behaved as a dithiothreitol oxidase, whose catalytic cycle was dependent on oxygen concentration. The oxidase cycle of glutathione was inhibited by mercaptosuccinate, while that of dithiothreitol was not affected. When mercaptosuccinate was substituted for GSH, a stable selenenylsulfide was formed. These observations suggest that electrostatic interactions affect the reductive cleavage of diselenide and selenenylsulfide linkages. This study illustrates the ease of one-electron transfers from RSe- to a variety of reducible substrates. Such free radical mechanisms may explain much of the cytotoxicity of alkylselenols, and they demonstrate that selenocystamine is a poor catalytic model of the enzyme glutathione peroxidase.     

Salt inactivation as a mechanistic probe of membrane-bound chloroplast coupling factor 1.

Conditions are reported under which ATP protects membrane-bound coupling factor 1 against sodium bromide inactivation. The presence of Mg2+ was found to be obligatory for this protection. ADP and GTP also protected the enzyme against salt inactivation but to a much smaller extent. Other nucleotides tested were ineffective. At low ATP concentrations ADP prevented the effect of ATP and modified the saturation curve for ATP from hyperbolic to sigmoidal. Treatment of chloroplasts with 0.4 M MgCl2 or 2 M LiCl resulted in inactivation of photophosphorylation. In contrast to NaBr-depleted particles the MgCl2 or LiCl-depleted chloroplasts can be reconstituted by purified coupling factor 1. A binding site for Mg2+ and two different sites for ATP upon the coupling factor 1 are suggested to explain the mechanism of their protection against salt inactivation.     

The reaction between nitrite and oxyhemoglobin: a mechanistic study

The nitrite anion (NO(-)(2)) has recently received much attention as an endogenous nitric oxide source that has the potential to be supplemented for therapeutic benefit. One major mechanism of nitrite reduction is the direct reaction between this anion and the ferrous heme group of deoxygenated hemoglobin. However, the reaction of nitrite with oxyhemoglobin (oxyHb) is well established and generates nitrate and methemoglobin (metHb). Several mechanisms have been proposed that involve the intermediacy of protein-free radicals, ferryl heme, nitrogen dioxide (NO(2)), and hydrogen peroxide (H(2)O(2)) in an autocatalytic free radical chain reaction, which could potentially limit the usefulness of nitrite therapy. In this study we show that none of the previously published mechanisms is sufficient to fully explain the kinetics of the reaction of nitrite with oxyHb. Based on experimental data and kinetic simulation, we have modified previous models for this reaction mechanism and show that the new model proposed here is consistent with experimental data. The important feature of this model is that, whereas previously both H(2)O(2) and NO(2) were thought to be integral to both the initiation and propagation steps, H(2)O(2) now only plays a role as an initiator species, and NO(2) only plays a role as an autocatalytic propagatory species. The consequences of uncoupling the roles of H(2)O(2) and NO(2) in the reaction mechanism for the in vivo reactivity of nitrite are discussed.     

Oxidation of melatonin by oxoferryl hemoglobin: a mechanistic study.

Reaction of melatonin with the hypervalent iron centre of oxoferryl hemoglobin, produced in aqueous solution from methemoglobin and H2O2, has been investigated at 37 degrees C and pH 7.4, by absorption spectroscopy. The reaction results in reduction of the oxoferryl moiety with formation of a heme-ferric containing hemoprotein. Stopped-flow spectrophotometric measurements provide evidence that the reduction of oxoferryl-Hb by melatonin is first-order in oxoferryl-Hb and first-order in melatonin. The bimolecular reaction constant at pH 7.4 and 37 degrees C is 112 +/- 1.0 M(-1) s(-1). Two major oxidation products from melatonin have been found by gas chromatography-mass spectroscopy: the cyclic compound 1,2,3,3a,8,8a-hexahydro-1-acetyl-5-methoxy-3a-hydroxypyrrolo[2,3-b]indole (cyclic 3-hydroxy-melatonin), and N-acetyl-N'-formyl 5-methoxykynuramine (AFMK). The percentage yield of the two major products appears dependent on the ratio [oxoferryl-Hb]:[melatonin]--the higher the ratio the higher the yield of AFMK. The observed stoichiometry oxoferryl-Hb(reduced):melatonin(consumed) is 2, when the ratio [oxoferryl-Hb]:[melatonin] is 1:1, but appears >2 at higher molar ratios. The reduction of the hypervalent iron of the oxoferryl moiety may be consistent with an oxidation of melatonin by two one-electron steps.     

Structural and mechanistic basis of bacterial sugar nucleotide-modifying enzymes

Recently, carbohydrates have come to the fore because of their central role in many biological processes. One area of current interest concerns the enzymatic modification of sugar nucleotides, in relation to both secondary metabolite glycosylation and the formation of complex cell surface-associated glycoconjugates. Bacteria, in particular, have proven to be a rich field in which to study these transformations, because they are often unique to specific classes of organisms. This has led to the realization that such microbial biosynthetic pathways might be exploited in the generation of novel antibiotics, or indeed serve as targets for such compounds. This work illustrates the interplay between protein structure determination, chemistry, and molecular biology in providing insight into the mechanism of such biochemical transformations.     

Catalase-like activity of human methemoglobin: a kinetic and mechanistic study

Hydrogen peroxide triggers a redox cycle between methemoglobin and ferrylhemoglobin, leading to protein inactivation and oxygen evolution. In the present paper, the catalase-like oxygen production by human methemoglobin in the presence of H₂O₂ was kinetically characterized with a Clark-type electrode. Progress curves showed a pseudo-steady state in the first minutes of the reaction, while double-reciprocal plots were upwardly concave, indicating positive co-operativity dependent upon protein concentration, which is a very unusual kinetic behavior. Addition of superoxide radical scavengers slightly increased activity, suggesting that most oxygen was produced biocatalytically. By considering all the experimental data obtained, a possible mechanism was proposed, including: (a) competition between the one-electron and the two-electron reductions of the oxoferryl free radical species of hemoglobin, giving rise to ferrylhemoglobin and methemoglobin, respectively; (b) competition between the superoxide-dependent inactivation of the protein and its reduction back to the met state. Computer simulations of that model have been performed by numerically integrating the differential equations set describing the mechanism, which was seen to yield predictions of the kinetic parameters variation consistently with the kinetic behavior experimentally observed. We suggest that the catalase-like activity of methemoglobin must predominantly be a biocatalytic reaction that protects the protein against H₂O₂-induced suicide inactivation.