Reversible modification of amino groups in aspartate aminotransferase.

Amino groups in the pyridoxal phosphate, pyridoxamine phosphate, and apo forms of pig heart cytoplasmic aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC .2.6.1.1) have been reversibly modified with 2,4-pentanedione. The rate of modification has been measured spectrophotometrically by observing the formation of the enamine produced and this rate has been compared with the rate of loss of catalytic activity for all three forms of the enzyme. Of the 21 amino groups per 46 500 molecular weight, approx. 16 can be modified in the pyridoxal phosphate form with less than a 50% change in the catalytic activity of the enzyme. A slow inactivation occurs which is probably due to reaction of 2,4-pentanedione with the enzyme-bound pyridoxal phosphate. The pyridoxamine phosphate enzyme is completely inactivated by reaction with 2,4-pentanedione. The inactivation of the pyridoxamine phosphate enzyme is not inhibited by substrate analogs. A single lysine residue in the apoenzyme reacts approx. 100 times faster with 2,4-pentanedione than do other amino groups. This lysine is believed to be lysine-258, which forms a Schiff base with pyridoxal phosphate in the holoenzyme.

Determination of buffering capacity of rat myocardium during ischemia.

To determine the buffering capacity of ischemic rat myocardium, lactate production was altered by glycogen depletion prior to total global ischemia. Lactate production was monitored by 1H-NMR spectroscopy in perfused rat hearts and determined by enzymatic assay of freeze-clamped tissue extracts. Intracellular pH was measured by 31P-NMR spectroscopy. The relationship between total lactate produced and pH varied considerably, depending on the final pH reached. At pH greater than 6.4 this relationship is linear with a total buffering capacity (delta lactate/delta pH) of 25 mumol H+/g wet weight per pH unit. At lower pH values (pH less than 6.4), the total buffering capacity increases progressively. Since ischemia is invariably accompanied by ATP and phosphocreatine (PCr) hydrolysis, the proton production/consumption during high-energy phosphate hydrolysis must be considered when evaluating the intrinsic buffering capacity of the myocardium against proton loads produced by lactate production from glucose and glycogen. Schemes are presented which allow an estimation of the contribution of ATP and PCr hydrolysis and the buffering by the CO2/HCO3- system during ischemia. At pH greater than 6.4, the majority (about 60%) of buffering is due to hydrolysis of adenosine triphosphate, phosphocreatine in the heart, and neutralization of sodium bicarbonate in the perfusate. At pH less than 6.4 an increasing proportion of cardiac buffering is from intrinsic cardiac buffers, most likely from intracellular proteins. After correction for these contributions to the observed total cardiac buffering capacity, the intrinsic buffering capacity of the myocardium can be accounted for by a high capacity (170 mumol/g wet weight) but low pKa (5.2) buffering system.

Protein chaperones and protein folding.

A universal strategy for obtaining maximal protein expression or refolding remains elusive; however, headway has been made toward understanding these processes in vivo. The observation of reversible protein aggregation, asymmetry in protein-chaperone complexes, redox effects on disulfide formation, and the sequential involvement of multiple chaperones and foldases may suggest new approaches. Such new approaches include immobilized catalysts and manipulation of the bacterial periplasm.

Inhibition of glucose phosphorylation by fatty acids in the perfused rat heart.

The flux of glucose entering the glycolytic pathway under various metabolic conditions has been indirectly monitored in the Langendorff perfused rat heart using 31P-NMR spectroscopy. By totally inhibiting (greater than 95%) glyceraldehyde-3-phosphate dehydrogenase with low concentrations of iodoacetic acid (0.2 mM) in the perfusion medium, active glycolysis results in the accumulation of sugar phosphate species (fructose 1,6-bisphosphate, dihydroxyacetone phosphate, and glyceraldehyde 3-phosphate) which can be observed in the 31P-NMR spectrum. Using this technique, it has been shown that butyrate (10 mM) in the perfusion medium decreases the flux through the initial steps of the glycolytic pathway by at least 6-fold and that both glucose phosphorylation and glycogenolysis are inhibited. Upon total global ischemia in the presence of both glucose and butyrate, the glycolysis rate is stimulated approx. 100-fold.

Facilitating the formation of disulfide bonds in the Escherichia coli periplasm via coexpression of yeast protein disulfide isomerase.

Sacchromyces cerevisiae protein disulfide isomerase (yPDI) was expressed in the E. coli periplasm by using plasmids encoding the OmpA-yPDI-(His)(6) fusion gene under the control of the araBAD, trc, or T7 promoter. The expression levels of yeast PDI under these promoters were compared. Our results showed that yeast PDI expressed into the periplasm could catalyze the formation of disulfide bonds in alkaline phosphatase, restoring the phoA(+) phenotype in dsbA(-) mutants. The yeast PDI was purified from the Escherichia coli periplasm and shown to exhibit catalytic properties comparable to those of the rat enzyme with reduced RNase as substrate. In vivo, coexpression of the yeast PDI increased the yield of bovine pancreatic trypsin inhibitor (BPTI) in E. coli by 2-fold, similar to the effect seen previously with the coexpression of the rat enzyme. However yeast PDI was more effective than rat PDI in facilitating the expression of active tissue plasminogen activator (tPA). These results point to differences in the substrate specificity of various PDI enzymes, at least in the context of the E. coli periplasm.

Biological disulfides: the third messenger? Modulation of phosphofructokinase activity by thiol/disulfide exchange.

Rabbit muscle phosphofructokinase is rapidly inactivated at pH 8.0 by incubation with low concentrations of oxidized glutathione, Coenzyme A glutathione mixed disulfide, and oxidized Coenzyme A. The inactivation is first order in disulfide concentration over the concentration ranges examined (50-200 microM), and is approximately 8-fold slower at pH 7.0 than at pH 8.0. The substrates ATP and fructose 6-phosphate protect against inactivation while effector molecules such as AMP, cAMP, and citrate do not. The oxidation of the enzyme by disulfides is fully reversible. The equilibrium constant for the reaction Ered + GSSG in equilibrium Eox + GSH at pH 8.0 is 7.1 in the absence of substrates and 2.5 in the presence of 0.1 mM ATP. For comparison, the equilibrium constant for the reaction CoASH + GSSG in equilibrium CoASSG + GSH was found to be 3.1 at pH 8.0. These equilibrium constants for thiol/disulfide exchange are such that modulation of phosphofructokinase activity by thiol/disulfide exchange in vivo is feasible. The ability of the thiol/disulfide ratio in vivo to modulate the activity of the fructose 6-phosphate/fructose 1,6-diphosphate futile cycle is discussed. The possibility is considered that modulation of the thiol/disulfide ratio in vivo may serve as a "third messenger" in response to cAMP levels, and that the activity of key enzymes of glycolysis/gluconeogenesis may be regulated in response to changing thiol/disulfide ratios.

Catalysis of thiol/disulfide exchange: single-turnover reduction of protein disulfide-isomerase by glutathione and catalysis of peptide disulfide reduction.

Protein disulfide-isomerase, a protein localized to the lumen of the endoplasmic reticulum of eukaryotic cells, catalyzes the posttranslational formation and rearrangement of protein disulfide bonds. As isolated from bovine liver, the enzyme contains 0.8 free sulfhydryl group per mole of protein monomer and 3.1 disulfide bonds. Single-turnover experiments in which the disulfide bonds of the native enzyme are reduced by glutathione reveal three distinct reduction steps corresponding to the sequential reduction of the three disulfide bonds. The fastest disulfide to be reduced undergoes a change in the rate-determining step with increasing GSH concentration from a step which is second-order with respect to GSH concentration to a step which is first-order in GSH concentration. The disulfide which is reduced at an intermediate rate displays kinetics that are first-order in GSH concentration, and the slowest disulfide to be reduced exhibits kinetics which are second-order in GSH concentration. The enzyme catalyzes the steady-state reduction of a disulfide-containing hexapeptide (CYIQNC) by GSH. Initial velocity kinetic experiments are consistent with a sequential addition of the substrates to the enzyme. Saturation behavior is not observed at high levels of both substrates (Km for GSH much greater than 14 mM, Km for CYIQNC much greater than 1 mM). Only one of the three disulfides appears to be kinetically competent in the steady-state reduction of CYIQNC by GSH. The second-order thiol/disulfide exchange reactions catalyzed by the enzyme are 400-6000-fold faster than the corresponding uncatalyzed reactions.

Thiol/disulfide exchange between 3-hydroxy-3-methylglutaryl-CoA reductase and glutathione. A thermodynamically facile dithiol oxidation.

In glutathione redox buffers, rat liver, microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase rapidly equilibrates between a reduced, active form and an oxidized, inactive form. At pH 7.0, 37 degrees C, the second order rate constant for inactivation of the reduced enzyme by GSSG is 1700 +/- 200 M-1 min-1, approximately 20-fold faster than the reaction of GSSG with a typical, unhindered thiol of pKa 7.7. High concentrations of GSH or lower concentrations of dithiothreitol restore the activity of the oxidized enzyme. The oxidation of the enzyme by GSSG is only 30-fold slower in the presence of saturating levels of both substrates. The incomplete inhibition of thiol/disulfide exchange by substrates can lead to significant changes in the activity of the enzyme during the assay when glutathione is present. At redox equilibrium, both in the absence and presence of substrates, the activity of the enzyme depends on the quantity [GSH]2/[GSSG], suggesting that the redox transition involves the formation of a protein-SS-protein disulfide. The equilibrium constant for the reaction HMGRred + GSSG in equilibrium HMGRox + 2 GSH is 0.55 +/- 0.07 M in the absence of substrates and 0.20 +/- 0.02 M in the presence of saturating levels of both substrates. Thus, HMG-CoA reductase is very sensitive to dithiol oxidation both kinetically and thermodynamically. Significant changes in the oxidation state and activity of this enzyme could be expected to result from normal changes in the thiol/disulfide oxidation state of the cellular glutathione redox buffer.

Examination of the role of thiolimidate formation in the cleavage of acetoacetyl-CoA catalyzed by thiolase I from porcine heart.

The potential contribution of thiolimidate formation to the increased kinetic acidity of the alpha-proton of acetyl-CoA in the carbon-carbon bond forming reaction catalyzed by 3-ketoacyl-CoA thiolase (thiolase I) from porcine heart was assessed by chemical modification and isotope exchange experiments. Thiolase is only partially inactivated after the chemical modification of lysine residues by reductive methylation, pyridoxal phosphate, or o-phthaldehyde (specific for vicinal lysine and cysteine). The thiolase-catalyzed formation of acetyl-CoA from acetoacetyl-CoA and CoASH in 18OH2 is not accompanied by the appearance of 18O in the acetyl-CoA product. These experiments effectively rule out participation of thiolimidate formation in the thiolase reaction. Other mechanisms must be employed to facilitate the abstraction of the alpha-proton of acetyl-CoA by thiolase I.

Hormone binding by protein disulfide isomerase, a high capacity hormone reservoir of the endoplasmic reticulum.

Protein disulfide isomerase (PDI) is a folding assistant of the eukaryotic endoplasmic reticulum, but it also binds the hormones, estradiol, and 3,3',5-triiodo-l-thyronine (T(3)). Hormone binding could be at discrete hormone binding sites, or it could be a nonphysiological consequence of binding site(s) that are involved in the interaction PDI with its peptide and protein substrates. Equilibrium dialysis, fluorescent hydrophobic probe binding (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS)), competition binding, and enzyme activity assays reveal that the hormone binding sites are distinct from the peptide/protein binding sites. PDI has one estradiol binding site with modest affinity (2.1 +/- 0.5 microm). There are two binding sites with comparable affinity for T(3) (4.3 +/- 1.4 microm). One of these overlaps the estradiol site, whereas the other binds the hydrophobic probe, bis-ANS. Neither estradiol nor T(3) inhibit the catalytic or chaperone activity of PDI. Although the affinity of PDI for the hormones estradiol and T(3) is modest, the high local concentration of PDI in the endoplasmic reticulum (>200 microm) would drive hormone binding and result in the association of a substantial fraction (>90%) of the hormones in the cell with PDI. High capacity, low affinity hormone sites may function to buffer hormone concentration in the cell and allow tight, specific binding to the true receptor while preserving a reasonable number of hormone molecules in the very small volume of the cellular environment.

Inactivation of hydroxymethylglutaryl-CoA reductase from yeast by coenzyme A disulfide.

The time-dependent inactivation of hydroxymethylglutaryl-CoA reductase from yeast by solutions of hydroxymethylglutaryl-CoA and CoASH is due to the rapid inactivation of the enzyme by oxidized CoA (CoA disulfide) present at trace levels in solutions of hydroxymethylglutaryl-CoA and CoASH. Solutions of hydroxymethylglutaryl-CoA or CoASH incubated for 1.5 h with 10 mM dithiothreitol at pH 7.0, 22 degrees C, do not inactivate the enzyme. Inactivation of hydroxymethylglutaryl-CoA reductase is rapid and complete at concentrations of CoA disulfide comparable to those measured in solutions of hydroxymethylglutaryl-CoA and CoASH. Inactivation of the enzyme by CoA disulfide may be reversed by treating the inactive enzyme with 10 mM dithiothreitol at pH 7.0 Both the inactivation of the enzyme by CoA disulfide and reactivation by dithiothreitol are inhibited by hydroxymethylglutaryl-CoA. Other disulfides such as Ellman's reagent and glutathione disulfide also inactivate the enzyme. A thio-disulfide exchange reaction with a sulfhydryl group on the enzyme forming a mixed disulfide or an intramolecular protein disulfide could account for the enzyme inactivation. The normal function of the sulfhydryl group involved in the inactivation of the enzyme is unknown.

Thiol/disulfide exchange in the thioredoxin-catalyzed reductive activation of spinach chloroplast fructose-1,6-bisphosphatase. Kinetics and thermodynamics.

Two kinetically and thermodynamically distinct thiol/disulfide redox changes are observed during the reversible thioredoxin fb-catalyzed reduction and oxidation of spinach chloroplast fructose-1,6-bisphosphatase by dithiothreitol. The two processes, which occur at different rates and with different equilibrium constants, can be observed independently in either the reduction (activation) or oxidation (inactivation) direction by assaying the enzyme activity at different magnesium and fructose-1,6-bisphosphate concentrations. The two processes, in both the reduction and oxidation directions, are kinetically zero-order in dithiothreitol concentration and first-order in thioredoxin fb concentration. The rate-limiting step in both directions is the reaction of fructose-1,6-bisphosphatase with thioredoxin. The more kinetically and thermodynamically favored reduction of fructose-1,6-bisphosphatase lowers the apparent Km for fructose-1,6-bisphosphate while the less favorable process lowers the Km for magnesium. Both of the thiol/disulfide redox changes reach equilibrium in redox buffers consisting of different ratios of reduced to oxidized dithiothreitol (Ered + DTTox in equilibrium Eox + DTTred). The equilibrium constants (Kox) are 0.12 +/- 0.02 and 0.39 +/- 0.08 for the fast and slow reduction processes at pH 8.0. The equilibrium constants for oxidation of the enzyme by glutathione disulfide (Ered + GSSG in equilibrium Eox + 2 GSH) can be estimated to be approximately 2400 and 7800 M, respectively. Thermodynamically the fructose-1,6-bisphosphatase/thioredoxin fb system is extremely sensitive to oxidation, comparable to disulfide bond formation in extracellular proteins.

Scanning and escape during protein-disulfide isomerase-assisted protein folding.

During oxidative protein folding, efficient catalysis of disulfide rearrangements by protein-disulfide isomerase is found to involve an escape mechanism that prevents the enzyme from becoming trapped in covalent complexes with substrates that fail to rearrange in a timely fashion. Protein-disulfide isomerase mutants with only a single active-site cysteine catalyze slow disulfide rearrangements and become trapped in a covalent complex with substrate. Escape is mediated by the second, more carboxyl-terminal cysteine at the active site. A glutathione redox buffer increases the kcat for single-cysteine mutants by 20-40-fold, but the presence of the second cysteine at the active site in the wild-type enzyme increases the kcat by over 200-fold. A model is developed in which kinetic scanning for disulfides of increasing reactivity is timed against an intramolecular clock provided by the second cysteine at the active site. This provides an alternative, more efficient mechanism for rearrangement involving the reduction and reoxidation of substrate disulfides.

The effects of NADPH and 3-hydroxy-3-methylglutaryl-CoA on the thiol/disulfide redox behavior of rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase.

Microsomal 3-hydroxy-3-methylglutaryl-CoA reductase isolated from the livers of rats fed a diet containing cholestyramine (HMGR-C) is oxidized to a protein-SS-protein disulfide via a thermodynamically favorable thiol/disulfide exchange in glutathione redox buffers which approach the normal in vivo redox poise. In the presence of either substrate (NADPH or 3-hydroxy-3-methylglutaryl-CoA), the equilibrium thiol/disulfide redox behavior of HMGR-C is substantially different than that observed in the absence of substrates or in the presence of both substrates. NADPH present during redox equilibrium in a glutathione redox buffer decreases the equilibrium constant for formation of the protein-SS-protein disulfide (Kox,i) from 0.55 +/- 0.07 M to 0.18 +/- 0.02 M and increases the Kox,m for formation of an inactive protein-SS-glutathione mixed disulfide from less than 1 to 6 +/- 1. The presence of 3-hydroxy-3-methylglutaryl-CoA during redox equilibrium has a similar effect, decreasing the Kox,i for protein-SS-protein disulfide formation to 0.10 +/- 0.02 M and increasing the Kox,m for protein-SS-glutathione mixed disulfide formation to 3.8 +/- 0.9. A three-state model is developed which describes the simultaneous accumulation of protein-SS-protein and protein-SS-glutathione mixed disulfides at redox equilibrium with glutathione redox buffers. Because of the different redox behavior of the free and substrate-liganded forms of the enzyme, addition of 3-hydroxy-3-methylglutaryl-CoA or NADPH to HMGR-C at redox equilibrium results in increased reduction and activation of the enzyme.

Effect of protein and peptide inhibitors on the activity of protein disulfide isomerase.

The protein disulfide isomerase catalyzed reduction of insulin by glutathione is inhibited by peptides of various length and amino acid composition. Peptide inhibitors are competitive against insulin and noncompetitive against GSH, consistent with a sequential rather than a double displacement mechanism. Peptides of unrelated primary sequence that do not contain cysteine inhibit the GSH-insulin transhydrogenase activity of PDI, and the affinity of these peptides toward the enzyme is largely dependent on the peptide length rather than composition, hydrophobicity, or charge. Cysteine-containing peptides are 4-8-fold better inhibitors than non-cysteine-containing peptides of the same length, suggesting a cysteine-specific component to the interaction with the enzyme. Oxidized insulin chain B also inhibits the oxidative folding of reduced ribonuclease in a glutathione redox buffer with an inhibition constant that is comparable to that observed for the inhibition of insulin reduction, suggesting a similar if not identical binding site for the catalysis of oxidative protein folding and the reduction of insulin.

Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: dependence of the rate on the composition of the redox buffer.

The velocity of the oxidative renaturation of reduced ribonuclease A catalyzed by protein disulfide isomerase (PDI) is strongly dependent on the composition of a glutathione/glutathione disulfide redox buffer. As with the uncatalyzed, glutathione-mediated oxidative folding of ribonuclease, the steady-state velocity of the PDI-catalyzed reaction displays a distinct optimum with respect to both the glutathione (GSH) and glutathione disulfide (GSSG) concentrations. Optimum activity is observed at [GSH] = 1.0 mM and [GSSG] = 0.2 mM. The apparent kcat at saturating RNase concentration is 0.46 +/- 0.05 mumol of RNase renatured min-1 (mumol of PDI)-1 compared to the apparent first-order rate constant for the uncatalyzed reaction of 0.02 +/- 0.01 min-1. Changes in GSH and GSSG concentration have a similar effect on the rate of both the PDI-catalyzed and uncatalyzed reactions except under the more oxidizing conditions employed, where the catalytic effectiveness of PDI is diminished. The ratio of the velocity of the catalyzed reaction to that of the uncatalyzed reaction increases as the quantity [GSH]2/[GSSG] increases and approaches a constant, limiting value at [GSH]2/[GSSG] greater than 1 mM, suggesting that a reduced, dithiol form of PDI is required for optimum activity. As long as the glutathione redox buffer is sufficiently reducing to maintain PDI in an active form [( GSH]2/[GSSG] greater than 1 mM), the rate acceleration provided by PDI is reasonably constant, although the actual rate may vary by more than an order of magnitude. PDI exhibits half of the maximum rate acceleration at a [GSH]2/[GSSG] of 0.06 +/- 0.01 mM.

Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: pre-steady-state kinetics and the utilization of the oxidizing equivalents of the isomerase.

At low concentrations of a glutathione redox buffer, the protein disulfide isomerase (PDI) catalyzed oxidative renaturation of reduced ribonuclease A exhibits a rapid but incomplete activation of ribonuclease, which precedes the steady-state reaction. This behavior can be attributed to a GSSG-dependent partitioning of the substrate, reduced ribonuclease, between two classes of thiol/disulfide redox forms, those that can be converted to active ribonuclease at low concentrations of GSH and those that cannot. With catalytic concentrations of PDI and near stoichiometric concentrations of glutathione disulfide, approximately 4 equiv (2 equiv of ribonuclease disulfide) of GSH are formed very rapidly followed by a slower formation of GSH, which corresponds to an additional 2 disulfide bond equiv. The rapid formation of RNase disulfide bonds and the subsequent rearrangement of incorrect disulfide isomers to active RNase are both catalyzed by PDI. In the absence of GSSG or other oxidants, disulfide bond equivalents of PDI can be used to form disulfide bonds in RNase in a stoichiometric reaction. In the absence of a glutathione redox buffer, the rate of reduced ribonuclease regeneration increases markedly with increasing PDI concentrations below the equivalence point; however, PDI in excess over stoichiometric concentrations inhibits RNase regeneration.