How fumarase recycles after the malate --> fumarate reaction. Insights into the reaction mechanism.

Recycling of yeast fumarase to permit repetition of its reaction chemistry requires two proton transfers and two conformational changes, in pathways that are different in detail but thematically similar in the two directions. In the malate --> fumarate direction, simple anions such as acetate accelerate the fumarate-off step producing E(H(f)), a fumarate-specific isoform that retains the C3R-proton of malate. Fumarate specificity is shown with S-2,3-dicarboxyaziridine, which is competitive vs fumarate and noncompetitive with malate as substrate. The steady-state level of E(H(f)), based on Kii (S-2,3-dicarboxyaziridine), is increased by D2O and decreased by imidazole acting as a general acid for conversion of E(H(f)) to E(H(f))H. E(H(f))H is fumarate-specific as shown by the inhibition pattern with ClO4-. The pKa of this step is approximately 7.25 based on the pH dependence of Kii (ClO4-). A conformational change occurs next as shown by high sensitivity of k(cat) but not k(cat)/Km, to the microviscosogen, glycerol, and change to a nonspecific isoform, E(H(mf))H, probably the same species formed in the fumarate --> malate direction from malate-specific intermediates by a different conformational change. Malate enters the cycle by reaction with E(H(mf))H and returns to E(m)H x malate after a second conformational change. When fumarate-off is slow, as in low anion medium, malate itself becomes an activator of malate --> fumarate. This effect occurs with changes in inhibition patterns suggestive of the bypass of the slow E(f) --> E(mf) conversion in favor of direct formation of E(mf) when free fumarate is formed. 3-Nitro-2-hydroxypropionate, a strong inhibitor of fumarase [Porter, D. J. T., and Bright, H. J. (1980) J. Biol. Chem. 255, 4772-4780] in its carbanion form, is competitive with both malate and fumarate. Therefore, 3-nitro-2-hydroxypropionic acid interacts with E(H(mf))H and not with E(m) or E(f) isoforms. Occurrence of two different conformational changes in the recycling process suggests that the reaction chemistry employs a two-step mechanism. The specificity of inhibition for E(H(mf))H is consistent with the expected intermediate of a carbanion mechanism, E(H)H x carbanion-. The proton transfers and conformational changes of recycling occur in the same sequence that is expected for this reaction chemistry. Several examples of ligand-activated conformational changes are reported.

Restructuring the active site of fumarase for the fumarate to malate reaction.

Changes in the active site of fumarase (yeast fumarase II) that occur when fumarate is converted to malate (E.F --> E.M) must be reversed for another cycle of reaction to take place. As shown here, recycling of the enzyme includes two proton transfers and one conformational change. These events, together with the M-off step, are variously rate-determining depending on the medium. In very low salt the release of M is limited by the conformational change. Thus, (V/Km)F decreases with increased viscosity, shown with glycerol. A variety of simple anions, such as Cl- at approximately 50 mM and F itself at low concentration, activate the dissociation of M. This nonspecific anion effect is the basis for the >4-fold apparent cooperative activation by substrate. The M-off step and the conformational change are independent and random-order events. Thus, even when M-off is made rapid the rate of recycling is inhibited by glycerol, which in 100 mM NaCl inhibits Vmax but not V/Km. The enzyme form that results when M is released is M-specific, Em. Thus mesotartarate, competitive toward M, is noncompetitive toward F. The slow conformational change required for recycling of Em is activated by Pi and chaotropic anions such as azide and thiocyanate, giving rise to a nonspecific intermediate, Emf (mesotartarate becomes competitive toward F and Britton's countertransport property disappears with these activators). Evidence is presented for the locations and rates of the two proton transfer steps required to complete the cycle.

Role of conformational change in the fumarase reaction cycle.

Activation of fumarase by high concentrations of either malate or fumarate, often referred to as negative cooperativity, can be explained without assuming additional sites of substrate action or subunit-subunit interactions. The following observations support a model based on a rate-dependent recycling of free enzyme through a sequence of conformational states that differ in substrate specificity and catalytic activity: (1) Displacement from equilibrium of a radiolabeled malate/fumarate probe is readily induced by moderate concentrations of either substrate. This phenomenon, called substrate-induced countertransport, indicates that the steady-state ratio of free enzyme forms is very dependent on substrate concentration. (2) Related to this, the back-labeling that can be observed with either 14C product with either substrate in the steady state is more rapid than expected for a single free enzyme state model. (3) Fumarate, more strongly than malate, shows competitive effects as a product. This may reflect a higher affinity of fumarate for an isoform that also reacts with malate. (4) P(i), an activator of fumarase at midrange substrate concentration, overcomes strong competitive inhibition by fumarate of the M-->F reaction and increases recycling as shown by its effect on counterflow. To the extent that these effects are due to buffer activation, they suggest that proton transfer between solvent and the enzyme site is important in determining the recycling rate. (5) Transaconitate, a competitive inhibitor, overcomes counterflow induced by either substrate, indicating that recycling events occur in the enzyme-transaconitate complex.

Proton transfer in catalysis by fumarase.

Using 3T[14C]malate it was possible to show intermolecular T-transfer to unlabeled fumarate. The rate of dissociation of ET derived from the malate was not rapid, only about as fast as required for KMcat. Because of the slow dissociation of ET derived from T-malate, the awkward complex ET-malate is readily formed. As shown by the effect of added malate on the partition of ET, otherwise captured by fumarate, ET.malate must be functional. Its rate of dissociation to E.M determines the V/Km value of malate. Hydrogen dissociation of the complex ET.F was linearly related to the concentration and basicity of the buffer provided, varying from < 10% to > 60% of the overall rate with alkyl phosphonates. Partition of EH.F to free malate or fumarate occurs in a ratio approximately 2:1 at both low and high buffer. This agrees well with the comparison of the equilibrium exchange rates: malate with [18O]water to malate with [14C]-fumarate [Hansen, J.N., Dinovo, E.C., & Boyer, P.D. (1969) J. Biol. Chem. 244, 6270-6279]. Therefore, the abstracted hydroxyl group is fully exchanged from the enzyme when the bound hydrogen and fumarate return to malate and must be much more accessible to the medium than the abstracted proton. The fact that buffer increases the rate of proton transfer to the medium in the central complex makes it appear that a proton relay connects the active site donor with a remote site that interfaces with the ultimate proton source, water.

Role of CO2 in proton activation by histidine decarboxylase (pyruvoyl).

The amino acid decarboxylases that use an intrinsic pyruvoyl cofactor have been viewed in terms of the pyridoxal-P paradigm whereby a Schiff base is formed between the enzyme-bound cofactor and the substrate, setting up a cation sink for electrons of the C alpha-CO2- bond, ejecting CO2, and the reversal of these steps with a proton with overall retention stereochemistry. With histidine decarboxylase (pyruvoyl) it is found that the presence of CO2 is required for T-exchange between histamine and water. Since the forward reaction including formation of the C-H bond does not require added CO2, it might be assumed that the CO2 that is formed in the fragmentation step is retained by the enzyme perhaps to assist in proton transfer. No such requirement for CO2 has been reported for the pyridoxal-P-dependent decarboxylases which are generally thought to liberate CO2 prior to proton transfer. In seeking a connection between bound CO2 and proton transfer in the histidine decarboxylase reaction, one is reminded of the carboxybiotin enzymes also known for an invariant stereochemistry of retention and for the requirement that the biotin be in the carboxylated form for H-exchange to occur. Perhaps the bound CO2 of histidine decarboxylase forms a carbamate by addition to Lys155 or to an amide group of the active site. The new carboxy group could then be the vehicle for protonating the carbon from which it originated, giving overall retention of the stereochemistry at the alpha-C.(ABSTRACT TRUNCATED AT 250 WORDS)

A ubiquitin C-terminal isopeptidase that acts on polyubiquitin chains. Role in protein degradation.

In the ubiquitin (Ub) pathway, proteins are ligated with polyUb chains and then are degraded by a 26 S protease complex. We describe an enzyme, called isopeptidase T, that acts on polyUb chains. It is a monomeric Ub-binding protein abundant in erythrocytes and reticulocytes. The activity of the isopeptidase is inhibited by iodoacetamide and Ub aldehyde. Treatment of the enzyme with Ub aldehyde increased its affinity for free Ub, indicating the existence of two different Ub-binding sites and cooperativity between the two sites. Isopeptidase T acts on polyUb-protein conjugates, but not on conjugates in which the formation of polyUb chains was prevented by the use of reductively methylated Ub or on abnormal polyUb chains formed with a mutant Ub that contains a Lys----Arg substitution at residue 48. The enzyme converts high molecular mass polyUb-protein conjugates to lower molecular mass forms with the release of free Ub, but not of free protein substrate. The lower molecular mass Ub-protein conjugate products are resistant to further action of the enzyme. Isopeptidase T stimulates protein degradation in a system reconstituted from purified enzyme components. The enzyme also stimulates the degradation of proteins ligated to polyUb chains by the 26 S protease complex. Preincubation of polyUb-protein conjugates with the isopeptidase did not much increase their susceptibility to proteolysis by the 26 S complex. On the other hand, preincubation of conjugates with the 26 S protease complex and ATP increased the release of free Ub upon further incubation with the isopeptidase. It thus seems that a role of this isopeptidase in protein breakdown is to remove polyUb chain remnants following the degradation of the protein substrate moiety by the 26 S complex.

Enolpyruvate: chemical determination as a pyruvate kinase intermediate.

Despite many studies suggesting the role of enolpyruvate as a bound intermediate in the pyruvate kinase reaction, direct evidence for it has been lacking. By use of a combination of chemical trapping and isolation of a derivative, significant amounts of enzyme-bound enolpyruvate have now been demonstrated. The method distinguishes enolpyruvate. It is based on reaction of bromine with enolpyruvate in acid, derivatization of formed bromopyruvate with thionitrobenzoate, and resolution by reversed-phase HPLC of the thioether derivative. As little as 10 pmol of the thioether derivative could be quantitated reliably. With this method, the internal equilibria, including the E.ATP.enolpyruvate intermediate, have been determined. Enzyme-enolpyruvate concentration was shown to be pH-dependent. Phosphoenolpyruvate also reacts with bromine to form bromopyruvate. To quantitate enolpyruvate specifically in a background of phosphoenolpyruvate, advantage was taken of phosphoenolpyruvate's much greater stability in acid. When bromide/was added 10 min after the acid quench, ketonization of enolpyruvate was complete, and only phosphoenolpyruvate was measured. Enolpyruvate is thus determined by difference between the bromopyruvate measured with and without delayed bromine addition.

A rate-determining proton relay in the pyruvate kinase reaction.

This study ascribes the large steady-state D2O isotope effect on kcat of pyruvate kinase (PEP + ADP----pyruvate + ATP) to the reprotonation of the product form of the enzyme for use in forming pyruvate. Previous tritium trapping experiments [Rose, I. A., & Kuo, D. J. (1989) Biochemistry 28, 9579-9585] with muscle pyruvate kinase showed that the proton used for ketonization of enolpyruvate is derived from an enzyme "pool" that contains three kinetically equivalent hydrogens that could be trapped in a nontritiated "chase" medium by high levels of ADP and PEP. The exchange of this pool with the medium was rapid in the free enzyme (approximately 1400 s-1), prior to addition of PEP, and apparently much less in the completed complex. The dissociation rate constant was determined by using the dissociation-competition equation koffT = K1/2kcat/Km, where kcat/Km is the steady-state parameter for PEP and K1/2 is the concentration of PEP required to trap 50% of the isotope that could be trapped. The present study shows that the competition constant, K1/2, is decreased by approximately 5-fold in D2O, the same effect see on kcat under conditions where kcat/Km, measured in the steady state, is not changed. The common effect of D2O on kcat in the steady state and koffT in pulse/chase suggests that the forward reaction rate is determined by hydrogen transfer to the enzyme. Further evidence indicates that the kinetically important proton in question is the proton used for ketonization of enolpyruvate, the substrate proton.(ABSTRACT TRUNCATED AT 250 WORDS)

Proton diffusion in the active site of triosephosphate isomerase.

The current model for hydrogen flow in the aldose-ketose isomerases is probably incorrect. Enzymes of this class are characterized by both hydrogen transfer and proton exchange in the interconversion of substrate and product. The transfer is believed to be due to the action of a unique basic residue in the active site. Exchange is presumed to occur by dissociation of the abstracted proton and reassociation from the medium prior to its transfer to the intermediate enediol on the way to product. Dissociation of a necessary proton from the intermediate state imposes limits on the overall catalytic rate depending on the pKa of the protonated base and the pH of the medium. A case in point is triose-P isomerase (TIM), where kcat is approximately 10(4) s-1. T-Labeled substrate is found to lose approximately 95% of its T to the medium when totally converted to product. Although the active site base is believed to be a glutamate of pKa = 3.9, the pH dependence of maximum velocity is known to be flat up to pH 10. The loss of hydrogen required to form product as indicated by isotope exchange must be restored completely at this high pH, requiring a base of very high pKa, or there must be some other explanation for the loss of isotope. The present study demonstrates the existence of a single proton on human and rabbit TIM and three protons on yeast TIM that rapidly exchange with the abstracted proton at the E.enediol state internal exchange. Exchange with the medium external exchange occurs from the enzyme after substrate or product has dissociated.(ABSTRACT TRUNCATED AT 250 WORDS)

The substrate proton of the pyruvate kinase reaction.

The pyruvate kinase reaction occurs in separate phosphate- and proton-transfer stages: (formula; see text) K+, Mg2+, and Mg.ADP are known to be required for the phosphoryl transfer step, and K+ and Mg2+ with allosteric stimulation by MgATP are important for proton transfer. This paper uses the isotope trapping method with 3H-labeled water to identify the proton donor and determine when in the sequence of the catalytic cycle it is generated. When the enzyme was allowed to exchange briefly with 3H2O (pulse phase) and then diluted into a mixture containing PEP, ADP, and the cofactor K+, Mg2+, or Co2+ in D2O (chase phase), an amount of [3H]pyruvate was formed in great excess of the amount expected from steady-state catalysis in the diluted 3H-labeled water. With K+, Mg2+, and ADP at pH 6-9.5 in the pulse phase, a limit of 1.25 enzyme equiv of 3H were trapped. The concentration of PEP required for half-maximum trapping was 14-fold greater than its steady-state Km. Therefore, the rate constant for dissociation of the donor proton is estimated to be 14 times the steady-state rate of [3H]pyruvate formation, approximately 109 s-1, or 1500 s-1. At pD 6.4, Mg2+ and ADP were required in the chase, indicating that the ADP in the pulse was not bound tightly enough to be used in the chase. At pD 9.4, ADP was not required in the chase, only Mg2+ or Co2+, making it possible to limit the chase to one turnover from hybrid labeled complexes such as E.K.Mg.CoADP or E.K.Co.MgADP and PEP.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of the glucose-1,6-bisphosphate system in brain and retina.

The distribution of glucose-1,6-bisphosphate (G16P2) synthase was measured in more than 70 regions of mouse brain, and nine layers of monkey retina. Activities in gray areas varied as much as 10-fold, in a hierarchical manner, from highest in telencephalon, especially the limbic system, to lowest in cerebellum, medulla, and spinal cord. The synthase levels were significantly correlated among different regions with G16P2 itself, as well as with previously published levels of a brain specific IMP-dependent G16P2 phosphatase. In contrast, neither G16P2 nor either its synthase or phosphatase correlated positively with phosphoglucomutase, and in all regions the G16P2 levels greatly exceeded requirements for activation of this mutase. This strengthens the view that G16P2 has some function besides serving as coenzyme for phosphoglucomutase. However, attempts to correlate the "G16P2 system," as defined by the three coordinately related elements, synthase, phosphatase, and G16P2, with other enzymes of carbohydrate metabolism, or with regional data of Sokoloff et al. [J. Neurochem. 28, 897-916 (1977)] for glucose consumption, were unsuccessful. This leaves open the possibility that brain G16P2 might serve as a phosphate donor for specific nonmetabolic effector proteins.

Aconitase: its source of catalytic protons.

An ordinary isotope partition experiment was performed to determine the rate of dissociation of the proton from the donor site for the hydration of cis-aconitate. Aconitase in [3H]water was efficiently diluted into well-mixed solutions of cis-aconitate. Citrate and isocitrate that were formed within 2 s were more heavily labeled than could be explained by consideration of an isotope effect in the processing of one proton per enzyme equivalent. Control experiments indicate that mixing was much more rapid than catalytic turnover, ruling out incompletely diluted [3H]water as a significant isotope source. Therefore, it appears that significantly more than one enzyme-bound tritium atom (protons) must have been used in the course of the multiple turnover of the enzyme after the dilution was complete. Isotope incorporation reached values in excess of four proton equivalents as a limit with simple Michaelis dependence on cis-aconitate. From the half-saturation concentration value for trapping, 0.15 mM, the t 1/2 for exchange of each of these protons with solvent appears to be approximately 0.1 s at 0 degrees C. The large number of protons trapped seems to suggest the existence of a structurally stabilized pool of protons, or water, that communicates between the active site base and the medium in the hydration of cis-aconitate. The proton abstracted in the dehydration of [3H]citrate is transferred directly to undissociated cis-aconitate to form isocitrate without dilution, or cis-aconitate having dissociated, the tritium passes to the medium, presumably through the pool of bound protons indicated above. All of the citrate-derived protons can be found in isocitrate if cis-aconitate is added in sufficient concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Ubiquitin-aldehyde: a general inhibitor of ubiquitin-recycling processes.

The generation and characterization of ubiquitin (Ub)-aldehyde, a potent inhibitor of Ub-C-terminal hydrolase, has previously been reported. We now examine the action of this compound on the Ub-mediated proteolytic pathway using the system derived from rabbit reticulocytes. Addition of Ub-aldehyde was found to strongly inhibit breakdown of added 125I-labeled lysozyme, but inhibition was overcome by increasing concentrations of Ub. The following evidence shows the effect of Ub-aldehyde on protein breakdown to be indirectly caused by its interference with the recycling of Ub, leading to exhaustion of the supply of free Ub: Ub-aldehyde markedly increased the accumulation of Ub-protein conjugates coincident with a much decreased rate of conjugate breakdown. release of Ub from isolated Ub-protein conjugates in the absence of ATP (and therefore not coupled to protein degradation) is markedly inhibited by Ub-aldehyde. On the other hand, the ATP-dependent degradation of the protein moiety of Ub conjugates, which is an integral part of the proteolytic process, is not inhibited by this agent. Direct measurement of levels of free Ub showed a rapid disappearance caused by the inhibitor. The Ub is found to be distributed in derivatives of a wide range of molecular weight classes. It thus seems that Ub-aldehyde, previously demonstrated to inhibit the hydrolysis of Ub conjugates of small molecules, also inhibits the activity of a series of enzymes that regenerate free Ub from adducts with proteins and intermediates in protein breakdown.

A specific endpoint assay for ubiquitin.

Simple endpoint assays for free ubiquitin (Ub) and for the Ub-activating enzyme are described. The method for measuring Ub makes use of the reaction of iodoacetamide-treated Ub-activating enzyme (E): [3H]ATP + Ub + E----E X [3H]AMP-Ub + PPi and PPi----2Pi (in the presence of pyrophosphatase). The Ub is then measured by determining the acid-insoluble radioactivity. The reaction is accompanied by a slow enzyme-catalyzed hydrolysis of the complex to AMP plus Ub. The presence of ubiquitin-activating enzyme in excess of Ub by approximately equal to 0.1 microM assures that the steady state will be close to the endpoint for total Ub. A preparation of the activating enzyme from human erythrocytes that does not depend on affinity chromatography is described. Several applications of the assay are presented.

Concentration and partitioning of intermediates in the fructose bisphosphate aldolase reaction. Comparison of the muscle and liver enzymes.

Chemical analysis of enzyme reaction intermediates has been used to compare the liver and muscle isozymes of rabbit aldolase at equilibrium and in their steady states to determine if they have properties that favor the direction of flow of glycolytic intermediates in their tissues of origin. For both enzymes at saturating concentrations of fructose 1,6-P2, the sum of intermediates in the steady state agreed with the total active enzyme calculated to be present. The two half-reactions, characterized by fructose 1,6-bisphosphate(Fru-P2):aldehyde exchange and DHAP:proton exchange were found to be of different importance in determining the rate of reaction with Fru-P2 with the liver enzyme being much more limited in the processing of DHAP. The chemical interconversions within each half-reaction are generally rapid compared with the release of products. The greater sensitivity of liver aldolase to inhibition by aldehydes in Fru-P2 cleavage seems to be a normal consequence of the higher level of the eneamine of DHAP in the forward steady state with the liver enzyme and probably should not be ascribed to a greater intrinsic affinity. An earlier report (Grazi, E., and Trombetta, G. (1979) Eur. J. Biochem. 100, 197-202) purporting to show a special interaction of glyceraldehyde-3-P with liver enzyme prior to proton abstraction from DHAP could not be reproduced. Examples are presented from the data that validate the use of the analytical methods used for analysis of intermediates in the case of the Schiff's base aldolases.

Mechanism of ubiquitin carboxyl-terminal hydrolase. Borohydride and hydroxylamine inactivate in the presence of ubiquitin.

Ubiquitin (Ub) carboxyl-terminal hydrolase (E) catalyzes the hydrolysis, at the Ub-carboxyl terminus, of a wide variety of C-terminal Ub derivatives. We show that the enzyme is inactivated by millimolar concentrations of either sodium borohydride or hydroxylamine, but only if Ub is present. We have interpreted these results on the assumption that the hydrolase mechanism is one of nucleophilic catalysis with an acyl-Ub-E intermediate. The borohydride-inactivated enzyme has the following properties. It is a stoichiometric complex of E and Ub containing tritium from sodium boro[3H]hydride. This complex is stable at neutral pH in 5 M urea and can be isolated on the basis of size on a sieving column, but a labeled product the size of Ub is released under more strongly denaturing conditions. The "Ub" released in acid is Ub-carboxyl-terminal aldehyde, based on the observations that: it contains the tritium present in the reduced complex and it is able to form the inactive enzyme from a stoichiometric amount of fresh enzyme, and inactivation is accompanied by E-Ub adduct formation; it has chemical properties expected of an aldehyde: after a second reduction of the Ub released with boro[3H]hydride and complete acid hydrolysis, tritium counts are found in ethanolamine (the carboxyl-terminal residue of Ub is glycine). These results suggest that enzyme and Ub combine in an equilibrium reaction to form an ester or thiol ester adduct (at the Ub-carboxyl terminus), and that this adduct is trapped by borohydride to give a very stable inactive E-Ub (thio) hemiacetal which is unable to undergo a second reduction step and which can release Ub-aldehyde in mild acid. Inactivation in the presence of hydroxylamine of hydrolase occurs once during hydrolysis of 1200 molecules of Ub-hydroxamate by the enzyme. The hydrolysis/inactivation ratio is constant over the range of 10-50 mM hydroxylamine showing that forms of E-Ub with which hydroxylamine and water react are different and not in rapid equilibrium. The inactive enzyme may be an acylhydroxamate formed from an E-Ub mixed anhydride generated from the E-Ub (thiol) ester inferred from the borohydride study. A direct radioactive assay for the hydrolase has been developed using the Ub-C-terminal amide of [3H]butanol-4-amine as substrate.

Ubiquitin carboxyl-terminal hydrolase acts on ubiquitin carboxyl-terminal amides.

Ubiquitin carboxyl-terminal hydrolase (formerly known as ubiquitin carboxyl-terminal esterase), from rabbit reticulocytes, has been shown to hydrolyze thiol esters formed between the ubiquitin carboxyl terminus and small thiols (e.g. glutathione), as well as free ubiquitin adenylate (Rose, I. A., and Warms, J. V. B. (1983) Biochemistry 22, 4234-4237). We now show that this enzyme hydrolyzes amide derivatives of the ubiquitin carboxyl terminus, including those of lysine (epsilon-amino), glycine methyl ester, and spermidine. It also hydrolyzes ubiquitin COOH-terminal hydroxamic acid, but is inactivated under the conditions for assaying ubiquitin-hydroxylamine adduct hydrolysis. Amide adducts formed between ubiquitin and epsilon-amino groups of protein lysine residues are much poorer substrates than is the ubiquitin amide of the epsilon-amino group of free lysine. The enzyme is thus a general hydrolase that recognizes the ubiquitin moiety, but is highly selective for small ubiquitin derivatives. It probably functions to regenerate ubiquitin from adventitiously formed ubiquitin amides and thiol esters. It also has the correct specificity to function in regenerating ubiquitin from small ubiquitin peptides that are probable end products of ubiquitin-dependent proteolysis. A simple, large-scale preparation of the enzyme from human erythrocytes is described.