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.

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.

Enzyme reactions of ATP studied by positional isotope exchange.

Reversible gamma-PO3 transfer in ATP reactions can be recognized by exchange of 18O from the beta,gamma-bridge position to the beta-P-nonbridge positions: (see article). Such intramolecular exchange is less demanding for the detection of the bond cleavage than the usual ATP:ADP isotope exchange because it does not require dissociation of bound ADP from the intermediate complex. Acyl phosphate intermediates are indicated for the glutamine synthetase and carbamyl-P synthetase reactions by their extreme requirements for glutamate and bicarbonate, respectively, for positional oxygen exchange. No support is given for E-P or concerted mechanisms. No support is found for an active CO2 in the latter reaction, although this is not ruled out by the data. Positional isomerization in ATP occurs with lamellae from spinach chloroplast only in the light. When the ATP molecule interacts, it also undergoes complete exchange of the gamma-PO3 oxygen with water before it rejoins the pool of free ATP. The difference in rates of the two exchanges suggests that the torsional motion of ADP-beta-PO3 is greatly hindered on the enzyme. This may explain, by the argument of substrate activation, the rapid reversibility of the ATPase reaction on the enzyme.

Chemistry of proton abstraction by glycolytic enzymes (aldolase, isomerases and pyruvate kinase).

Intermediates have been synthesized that are rapidly utilized by triose phosphate isomerase, yeast aldolase and pyruvate kinase. In each case the compounds have the properties of an enol expected for a stepwise proton transfer mechanism. Apparently the apparatus required for doing this chemistry is sufficiently unique for a large measure of structural homology to have been imposed upon the enzymes of this class during evolution.

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.

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)

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)

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 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.

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.

An enzyme with ubiquitin carboxy-terminal esterase activity from reticulocytes.

Thiols such as dithiothreitol (DTT) are known to allow recycling of the ubiquitin activating enzyme presumably due to attack by thiol on E-ubiquitin forming E + DTT-ubiquitin. It is now reported that the resulting ubiquitin thiol ester is extremely susceptible to hydrolysis, giving rise to free ubiquitin that can then also recycle in the activating enzyme reaction. The instability of ubiquitin thiol esters in this system is caused by a ubiquitin carboxy-terminal thiolesterase activity found as a minor contaminant of the activating enzyme. This activity of rabbit reticulocytes has been extensively purified, and some of its properties are reported. The enzyme, which also cleaves carboxy-terminal adenosine 5'-phosphate-ubiquitin, is inhibited by free ubiquitin at micromolar concentrations. The ubiquitin-specific esterase probably functions to hydrolyze glutathione and other thiol esters of ubiquitin that would be formed spontaneously from activated ubiquitin in cells.

Glucose exchange and catalysis by two crystalline hexokinase x glucose complexes. Evidence for an obligatory ATP-dependent conformational change in catalysis.

Hexokinase PI x glucose crystals grown with radiolabeled glucose under conditions similar to those used for x-ray diffraction studies (Bennett, W.S., Jr., and Steitz, T.A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4848-4852) have been shown to contain 1 mol of tightly bound glucose. These crystals exchange all of this glucose in a single exponential process (kobs = 0.7 min-1). In the crystalline form they are, however, inactive and do not catalyze formation of any bound glucose-6-P, suggesting that lattice forces prevent catalysis. A new catalytically active E x glucose complex has been crystallized in the presence of glucose and ADP, a competitive inhibitor of ATP. These crystals readily lose ADP upon washing with concentrated (NH4)2SO4. They exchange all of the tightly bound glucose more slowly than the form grown in the absence of ADP (kobs = 0.05 min-1). Addition of MgATP to the suspension in ammonium sulfate results in rapid conversion of half of the bound glucose to bound glucose 6-phosphate followed by further reaction as products are released. This agrees with the previously measured equilibrium constant of unity for enzyme-bound phosphoryl transfer catalyzed in solution (Wilkinson, K.D., and Rose, I.A. (1979) J. Biol. Chem. 254, 12567-12572). These results indicate that the two E x glucose crystals are distinguished by a nucleotide-dependent conformational difference, which is stabilized by lattice forces. The active crystals allow the facile dissociation of the ADP used to induce the change. This conformational change appears to be pevented in the E x glucose crystals and to be necessary to produce the active ternary complex.

Mechanism for oxygen exchange in the chloroplast photophosphorylation system.

The oxygen exchange that occurs between water and the gamma-PO3 of ATP in light-activated chloroplast lamellae was found to proceed with close to full equilibration of the oxygens before ATP returned to the medium. This is in contrast to the entry of approximately one water oxygen when ATP is synthesized from ADP and P1 in the same system. In the latter case, the limitation is kinetic, however, not steric, as shown by the presence of some molecules containing more than one water-derived oxygen in the gamma-PO3. The different extents of exchange can be explained by a relatively faster rate of dissociation of ATP from the chloroplast coupling factor during synthesis from ADP and P1 relative to its dissociation in the absence of net phosphorylation. To determine the mechanism of gamma-PO3:H2O exchange, its rate was compared with the rate of reversible cleavage of ATP as detected by betagamma bridge to beta nonbridge 18O scrambling in [Pbeta-18O-Pgamma]ATP (Midelfort, C. F., and Rose, I. A. (1976) J. Biol. Chem. 251, 5881-5887). The scrambling reaction, which depends on cleavage of the PbetaO--Pgamma bond, was found to occur in nearly the same fraction of ATP molecules that experienced gamma-PO3:H2O exchange in the same incubation, suggesting that the latter is due to multiple cycles of reversible ATP hydrolysis on the chloroplast coupling factor, i.e. [ATP-H2O in equilibrium ADP-Pi].

A stereochemical method for detection of ATP terminal phosphate transfer in enzymatic reactions. Glutamine synthetase.

An isotope scrambling method is described for the detection of transient [Enz:ADP:P-X] formation from [18O]ATP in ATP-coupled enzyme reactions. The method makes use of torsional symmetry of the newly formed (see article) group in ADP. [18 O]ATP labeled in the betagama bridge oxygen was incubated with enzyme and reversible cleavage of the PbetaO -- Pgamma bond was detected by the appearance of 18O in the beta nonbridge oxygens of the ATP pool. Experiments with sheep brain and Escherichia coli glutamine synthetases show that cleavage of ATP of enzyme-bound ADP and P-X requires glutamate. The exchange catalyzed by the E. coli enzyme with glutamate occurs in the absence of ammonia and is partially inhibited by added NH4Cl, as expected if the exchange is in the mechanistic pathway for glutamine synthesis. The results provide kinetic support for a two-step mechanism where phosphoryl transfer from ATP to glutamate precedes reaction with ammonia.