New probes of the F1-ATPase catalytic transition state reveal that two of the three catalytic sites can assume a transition state conformation simultaneously

MgADP in combination with fluoroscandium (ScFx) is shown to form a potently inhibitory, tightly bound, noncovalent complex at the catalytic sites of F(1)-ATPase. The F(1).MgADP.ScFx complex mimics a catalytic transition state. Notably, ScFx caused large enhancement of MgADP binding affinity at both catalytic sites 1 and 2, with little effect at site 3. These results indicate that sites 1 and 2 may form a transition state conformation. A new direct optical probe of F(1)-ATPase catalytic transition state conformation is also reported, namely, substantial enhancement of fluorescence emission of residue beta-Trp-148 observed upon binding of MgADP.ScFx or MgIDP. ScFx. Using this fluorescence signal, titrations were performed with MgIDP.ScFx which demonstrated that catalytic sites 1 and 2 can both form a transition state conformation but site 3 cannot. Supporting data were obtained using MgIDP-fluoroaluminate. Current models of the MgATP hydrolysis mechanism uniformly make the assumption that only one catalytic site hydrolyzes MgATP at any one time. The fluorometal analogues demonstrate that two sites have the capability to form the transition state simultaneously.     

Conserved walker A Ser residues in the catalytic sites of P-glycoprotein are critical for catalysis and involved primarily at the transition state step

P-glycoprotein mutants S430A/T and S1073A/T, affecting conserved Walker A Ser residues, were characterized to elucidate molecular roles of the Ser and functioning of the two P-glycoprotein catalytic sites. Results showed the Ser-OH is critical for MgATPase activity and formation of the normal transition state, although not for initial MgATP binding. Mutation to Ala in either catalytic site abolished MgATPase and transition state formation in both sites, whereas Thr mutants had similar MgATPase to wild-type. Trapping of 1 mol of MgADP/mol of P-glycoprotein by vanadate, shown here with pure protein, yielded full inhibition of ATPase. Thus, congruent with previous work, both sites must be intact and must interact for catalysis. Equivalent mutations (Ala or Thr) in the two catalytic sites had identical effects on a wide range of activities, emphasizing that the two catalytic sites function symmetrically. The role of the Ser-OH is to coordinate Mg(2+) in MgATP, but only at the stage of the transition state are its effects tangible. Initial substrate binding is apparently to an "open" catalytic site conformation, where the Ser-OH is dispensable. This changes to a "closed" conformation required to attain the transition state, in which the Ser-OH is a critical ligand. Formation of the latter conformation requires both sites; both sites may provide direct ligands to the transition state.     

Multinuclear magnetic resonance studies on serine protease transition state analogues

31P Nuclear Magnetic Resonance (NMR) studies were performed on mono- and diisopropylphosphoryl derivatives of alpha-chymotrypsin, trypsin, and subtilisin. Questions addressed included the pKa of the active center Asp...His...Ser triad in both species. While the pKa in the diisopropylphosphoryl derivatives is near 7.4 (found in this and other laboratories earlier) and reflects a nearly normal imidazolium titration curve, the apparent pKa in the monoisopropylphosphoryl enzymes (obtained by "aging" of the diisopropylphosphoryl derivatives and monitored by 31P NMR) is between 9.7 and 11.4 depending on the protease. This latter "titration" of the 31P NMR signal is reversible and presumably reflects the interaction of the imidazolium positive charge with the monoanionic phosphodiester. Of the two tetrahedral intermediates, the properties of the monoisopropylphosphoryl enzyme are probably more representative of the tetrahedral oxyanionic intermediate invoked during peptide hydrolysis. The same NMR technique was used to determine the action of PAM (pyridine-2-aldoxime methiodide, a known "antidote" for acetylcholinesterase inactivated by diisopropylfluorophosphate), on the inactivated enzymes. It was clear that the "antidote" could reverse the diisopropylphosphorylation but was ineffective on the monoisopropylphosphoryl ("aged") enzyme. 11B NMR studies were performed on phenylboronic (PBA) acid and 3,5-bis-trifluoromethylphenylboronic acid in the absence and presence of chymotrypsin and subtilisin. At 22 degrees C the former, but not the latter, compound was in fast exchange between the free and enzyme bound states. The relaxation parameters could be calculated for the bound PBA in chymotrypsin and the fluorinated analogue in subtilisin and clearly indicated that the boron nucleus was tetrahedral in the active centers, a good analogue for the tetrahedral oxyanionic intermediate.     

Steady state kinetics of enzymes with substrates having multiple reaction sites

An enzyme catalysing the reaction of a substrate with multiple reaction sites may display steady state kinetics described by a Michaels-Menten equation. The Km is identical for all sites considered individually and all sites together. The maximum velocity for a single site depends on the rate constants for reaction at that site and at all of other sites.     

A crystallographic study of the complex of phosphoramidon with thermolysin. A model for the presumed catalytic transition state and for the binding of extended substrates

The structure of the thermolysin inhibitor phosphoramidon (N-(α-l-rhamnopyranosyl-oxyhydroxyphosphinyl)-l-leucyl-l-tryptophan bound to the crystalline enzyme has been determined to a resolution of 2.3 Å by X-ray crystallography. The study shows that the complex of phosphoramidon with thermolysin resembles that of the presumed catalytic transition state inferred from the geometry of binding of dipeptide inhibitors. Also, the study reveals the mode of binding of thermolysin substrates extended on the imino side of the scissile peptide bond.The crystallographic results are consistent with a variety of other studies on the catalytic activity of thermolysin, and suggest a mechanism of action which is analogous to one of the two alternative mechanisms proposed by Lipscomb and co-workers (1968) for carboxypeptidase A. Key features of the proposed mechanism are that the substrate is initially bound to the enzyme with the carbonyl oxygen of the scissile peptide liganded to the zinc; that Glu143 promotes the nucleophilic attack of a buried water molecule on the carbonyl carbon, forming a tetrahedral intermediate; and that His231 acts as a proton donor. The observed binding of phosphoramidon to thermolysin provides further evidence supporting the mechanism in which Glu143 acts as a general base, promoting the attack of water on the carbonyl carbon, rather than the alternative mechanism in which Glu143 attacks the carbonyl carbon directly, forming an anhydride intermediate.     

Polyene substrates with unusual methylation patterns to probe the active sites of three catalytic antibodies

The synthesis of two tetraenes that differ in their methylation pattern from the natural substrate in lanosterol biosynthesis, 2,3-oxidosqualene, and their examination with three catalytic antibodies is described. The design of these novel, linear terpenoid structures was governed by initial results obtained from the characterization of the three catalytic antibodies. These were generated by immunization with a steroidal hapten that mimics multicyclization without the necessity for anti-Markovnikov additions or ring expansions. Such a reaction cascade would represent a more 'primitive' version compared to the oxidosqualene cyclization observed in lanosterol, cycloartenol and beta-amyrin biosynthesis and would not require a tail-to-tail connection of the third and fourth isoprene unit as seen in squalene. The first tetraene design (A) only contains trisubstituted double bonds and hence its synthesis starts from farnesol and tris-norgeraniol. The second tetraene design (B) is considered the more precise match to the inducing hapten that generated the antibody collections by exhibiting one disubstituted double bond and its synthesis utilizes a tris-norgeraniol derivative and a symmetrical bis-allylic alcohol as key building blocks. Chromatographic comparison studies lead to the conclusion that the currently studied antibodies also produce monocyclic products from the two substrates as has been formerly observed with a squalene-derived substrate. In contrast, 2,3-oxidosqualene is not accepted by these catalysts supporting the notion that the current substrates are fully bound by recognition of both terminal functional groups.     

Substrate variants versus transition state analogues as noncovalent reversible enzyme inhibitors

Reversible inhibitors are associated with fewer side effects than covalently binding ones and are, therefore, advantageous for treatment of conditions involving endogenous enzymes. Transition state analogue structures provide one design paradigm for such inhibitors; this paradigm seeks to exploit the capability of an enzyme active site to stabilise a transition state or associated intermediate. In contrast, structures that retain the functionality, and scissile bond of the substrate, can also act as reversible inhibitors; these are referred to here as substrate variants to distinguish them from substrate analogues. Their mode of inhibition depends on destabilisation of a reaction-path transition state or states. As the mode of destabilisation can be quite varied the scope to exploit substrate variants as reversible inhibitors is substantial. The two design paradigms are contrasted here and the case of substrate variants is delineated with a well-defined set of structures. These include the naturally occurring polypeptides BPTI (an inhibitor of a serine-based protease) and cathepsin propeptides (inhibitors of cysteine-based proteases) as well as the synthetic small-molecules cilastatin (an amide inhibitor of a zinc-based protease) and substituted mono- and tripeptides as inhibitors of cathepsins K and L.     

Conformations and arrangement of substrates at active sites of ATP-utilizing enzymes

The phosphoryl transferring enzymes pyruvate kinase, cAMP-dependent protein kinase and the pyrophosphoryl transferring enzyme PP-Rib-P synthetase utilize the beta, gamma bidentate metal--ATP chelate (delta-isomer) as substrate, as determined with substitution-insert CrIIIATP or CoIII(NH3)4ATP complexes. In addition, these enzymes bind a second divalent cation, which is an essential activator for pyruvate kinase and PP-Rib-P synthetase and an inhibitor of protein kinase. The enzyme-bound metal has been used as a paramagnetic reference point in T1 measurements to determine distances to the protons and phosphorus atoms of the bound nucleotide and acceptor substrates. These distances have been used to construct models of the conformations of the bound substrates. The activating metal forms a second sphere complex of the metal-nucleotide substrate on pyruvate kinase and PP-Rib-P synthetase while the inhibitory metal directly coordinates the polyphosphate chain of the metal-nucleotide substrate on protein kinase. Essentially no change is found in the dihedral angle at the glycosidic bond of ATP upon binding to pyruvate kinase (chi = 30 degrees), an enzyme of low base specificity, but significant changes in the torsional angle of ATP occur on binding to protein kinase (chi = 84 degrees) and PP-Rib-P synthetase (chi = 62 degrees), enzymes with high adenine-base specificity. Intersubstrate distances, measured with tridentate CrATP or beta, gamma bidentate CrAMPPCP as paramagnetic reference points, have been used to deduce the distance along the reaction coordinate on each enzyme. The reaction coordinate distances on pyruvate kinase (# +/- 1 A) and PP-Rib-P synthetase (not less than 3.8 A) are consistent with associative mechanisms, while that on protein kinase (5 +/- 0.7 A) allows room for a dissociative mechanism.     

Development of transition state analogues of purine nucleoside phosphorylase as anti-T-cell agents

Newborns with a genetic deficiency of purine nucleoside phosphorylase (PNP) are normal, but exhibit a specific T-cell immunodeficiency during the first years of development. All other cell and organ systems remain functional. The biological significance of human PNP is degradation of deoxyguanosine, and apoptosis of T-cells occurs as a consequence of the accumulation of deoxyguanosine in the circulation, and dGTP in the cells. Control of T-cell proliferation is desirable in T-cell cancers, autoimmune diseases, and tissue transplant rejection. The search for powerful inhibitors of PNP as anti-T-cell agents has culminated in the immucillins. These inhibitors have been developed from knowledge of the transition state structure for the reactions catalyzed by PNP, and inhibit with picomolar dissociation constants. Immucillin-H (Imm-H) causes deoxyguanosine-dependent apoptosis of rapidly dividing human T-cells, but not other cell types. Human T-cell leukemia cells, and stimulated normal T-cells are both highly sensitive to the combination of Imm-H to block PNP and deoxyguanosine. Deoxyguanosine is the cytotoxin, and Imm-H alone has low toxicity. Single doses of Imm-H to mice cause accumulation of deoxyguanosine in the blood, and its administration prolongs the life of immunodeficient mice in a human T-cell tissue xenograft model. Immucillins are capable of providing complete control of in vivo PNP levels and hold promise for treatment of proliferative T-cell disorders.     

Modelling transition state analogues and enzyme-inhibitor complexes of zinc-containing class II aldolases and metalloproteases

The zinc complex Tp^*Zn---OH (1:Tp^* = hydrotris-(3-cumyl,5-methyl-pyrazolyl)borate) which is a model of hydrolytic zinc enzymes was reacted with hydroxamic acids and hydroxyketones. Two hydroxamates Tp^*Zn-Hya (2: Hya = acetohydroxamate, 3: Hya = 2-hydroxamato-4-methylpentanoyl-alanyl-glycylamide) and two ketoalcoholates Tp^*Zn-Kea (4: Kea = hydroxyacetonate, 5: Kea = cumoylacetonate) were obtained. Crystal structure determinations of 2, 3 and 5 have revealed distorted ZnN₃O₂ coordinations in each case. The immediate environment of the zinc ion in the hydroxamates closely resembles that in enzyme-(hydroxamate)inhibitor complexes of zinc-containing metalloproteases like collagenases and thermolysin or of class II aldolases like fuculose-1-phoshate aldolase. Like in the enzymes the hydroxamates and the ketoalcoholates can serve as transition state analogues of the enzyme-catalysed reactions.     

Ionizable side chains at catalytic active sites of enzymes

Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1,072 Å³. The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes.     

Ionic changes in the mitotic apparatus at the metaphase/anaphase transition

We have employed a series of permeant, nontoxic, fluorescent probes to detect changes in ionic conditions within the mitotic apparatus of living endosperm cells of Haemanthus during the transition from metaphase to anaphase. Fluorescence emission intensity measurements from the spindle for chlorotetracycline (CTC) decline before the onset of anaphase, indicating a reduction in the amount of membrane- associated Ca2+ and suggesting an efflux of Ca2+ from membrane compartments into the spindle. Subsequent to the onset of anaphase, we observe increases in fluorescence with both 8-anilino-1-naphthalene sulfonate (ANS) and 3,3'-dipentyl 2,2'-dioxacarbocyanine (diO-C5(3)), sensitive to cationic and anionic charges at membrane surfaces, respectively. The increases with ANS and diO-C5(3) suggest that redistributions of ions within the spindle accompany anaphase motion. During the metaphase/anaphase transition, spindle membrane content remains constant, as evidenced by unchanging fluorescence with the hydrophobic probe, N-phenyl-1-naphthylamine (NPN). Shifts in emission intensity from the nonspindle cytoplasm or from the spindle poles do not accompany the changes in fluorescence we observe in the spindle, suggesting that any ionic fluxes responsible for the changes in fluorescence are restricted to the spindle domain.     

Over-the-barrier transition state analogues and crystal structure with Mycobacterium tuberculosis purine nucleoside phosphorylase

Stable chemical analogues of enzymatic transition states are imperfect mimics since they lack the partial bond character of the transition state. We synthesized structural variants of the Immucillins as transition state analogues for purine nucleoside phosphorylase and characterized them with the enzyme from Mycobacterium tuberculosis (MtPNP). PNPs form transition states with ribooxacarbenium ion character and catalyze nucleophilic displacement reactions by migration of the cationic ribooxacarbenium carbon between the enzymatically immobilized purine and phosphate nucleophiles. As bond-breaking progresses, carbocation character builds on the ribosyl group, the distance between the purine and the carbocation increases, and the distance between carbocation and phosphate anion decreases. Transition state analogues were produced with carbocation character and increased distance between the ribooxacarbenium ion and the purine mimics by incorporating a methylene bridge between these groups. Immucillin-H (ImmH), DADMe-ImmH, and DADMe-ImmG mimic the transition state of MtPNP and are slow-onset, tight-binding inhibitors of MtPNP with equilibrium dissociation constants of 650, 42, and 24 pM. Crystal structures of MtPNP complexes with ImmH and DADMe-ImmH reveal an ion-pair between the inhibitor cation and the nucleophilic phosphoryl anion. The stronger ion-pair (2.7 A) is found with DADMe-ImmH. The position of bound ImmH resembles the substrate side of the transition state barrier, and DADMe-ImmH more closely resembles the product side of the barrier. The ability to probe both substrate and product sides of the transition state barrier provides expanded opportunities to explore transition state analogue design in N-ribosyltransferases. This approach has resulted in the highest affinity transition state analogues known for MtPNP.     

Evidence for the selective population of FeMo cofactor sites in MoFe protein and its molecular recognition by the Fe protein in transition state complex analogues of nitrogenase

We have collected synchrotron x-ray solution scattering data for the MoFe protein of Klebsiella pneumoniae nitrogenase and show that the molecular conformation of the protein that contains only one molybdenum per alpha(2)beta(2) tetramer is different from that of the protein that has full occupancy i.e. two molybdenums per molecule. This structural finding is consistent with the existence of MoFe protein molecules that contain only one FeMo cofactor site occupied and provides a rationale for the 50% loss of the specific activity of such preparations. A stable inactive transition state complex has been shown to form in the presence of MgADP and AlF(4)(-). Gel filtration chromatography data show that the MoFe protein lacking a full complement of the cofactor forms initially a 1:1 complex before forming a low affinity 1:2 complex. A similar behavior is found for the MoFe protein with both cofactors occupied, but the high affinity 1:2 complex is formed at a lower ratio of Fe protein/MoFe protein. The 1:1 complex, MoFe protein-Fe protein x (ADP x AlF(4)(-))(2), formed with MoFe protein that lacks one of the cofactors, is stable. X-ray scattering studies of this complex have enabled us to obtain its low resolution structure at approximately 20-A resolution, which confirms the gel filtration finding that only one molecule of the Fe protein binds the MoFe protein. By comparison with the low resolution structure of purified MoFe protein that contains only one molybdenum per tetramer, we deduce that the Fe protein interacts with the FeMo cofactor-binding alpha-subunit of the MoFe protein. This observation demonstrates that the conformation of the alpha-subunit or the alpha beta subunit pair that lacks the FeMo cofactor is altered and that the change is recognized by the Fe protein. The structure of the 1:1 complex reveals a similar change in the conformation of the Fe protein as has been observed in the low resolution scattering mask and the high resolution crystallographic study of the 1:2 complex where both cofactors are occupied and with the Fe protein bound to both subunits. This extensive conformational change observed for the Fe protein in the complexes is, however, not observed when MgATP or MgADP binds to the isolated Fe protein. Thus, the large scale conformational change of the Fe protein is associated with the complex formation of the two proteins.     

Inhibition and structure of Trichomonas vaginalis purine nucleoside phosphorylase with picomolar transition state analogues

Trichomonas vaginalis is a parasitic protozoan purine auxotroph possessing a unique purine salvage pathway consisting of a bacterial type purine nucleoside phosphorylase (PNP) and a purine nucleoside kinase. Thus, T. vaginalis PNP (TvPNP) functions in the reverse direction relative to the PNPs in other organisms. Immucillin-A (ImmA) and DADMe-Immucillin-A (DADMe-ImmA) are transition state mimics of adenosine with geometric and electrostatic features that resemble early and late transition states of adenosine at the transition state stabilized by TvPNP. ImmA demonstrates slow-onset tight-binding inhibition with TvPNP, to give an equilibrium dissociation constant of 87 pM, an inhibitor release half-time of 17.2 min, and a Km/Kd ratio of 70,100. DADMe-ImmA resembles a late ribooxacarbenium ion transition state for TvPNP to give a dissociation constant of 30 pM, an inhibitor release half-time of 64 min, and a Km/Kd ratio of 203,300. The tight binding of DADMe-ImmA supports a late SN1 transition state. Despite their tight binding to TvPNP, ImmA and DADMe-ImmA are weak inhibitors of human and P. falciparum PNPs. The crystal structures of the TvPNP x ImmA x PO4 and TvPNP x DADMe-ImmA x PO4 ternary complexes differ from previous structures with substrate analogues. The tight binding with DADMe-ImmA is in part due to a 2.7 A ionic interaction between a PO4 oxygen and the N1' cation of the hydroxypyrrolidine and is weaker in the TvPNP x ImmA x PO4 structure at 3.5 A. However, the TvPNP x ImmA x PO4 structure includes hydrogen bonds between the 2'-hydroxyl and the protein that are not present in TvPNP x DADMe-ImmA x PO4. These structures explain why DADMe-ImmA binds tighter than ImmA. Immucillin-H is a 12 nM inhibitor of TvPNP but a 56 pM inhibitor of human PNP. And this difference is explained by isotope-edited difference infrared spectroscopy with [6-18O]ImmH to establish that O6 is the keto tautomer in TvPNP x ImmH x PO4, causing an unfavorable leaving-group interaction.     

Mapping transition states of protein unfolding by protein engineering of ligand-binding sites.

We describe a method for probing the integrity and relative orientation of structural elements that are indirectly linked by ligands in protein complexes during protein folding. The effect of 3'-GMP on the rate constants of unfolding of wild-type barnase and several mutants has been studied. By comparing the rates of unfolding of wild-type and mutant proteins, we show that the interaction between His102 and 3'-GMP is fully retained in the transition state compared with the folded state, while the interaction between Glu60 and the ligand is partly retained and that of Lys27 is broken. Our data suggest that the transition state has a partly formed ligand binding site in which the guanine binding loop containing Glu60 and the loop containing His102 are formed at the sides of the beta-sheet but the docking of the N terminus of the second alpha-helix containing Lys27 on the beta-sheet is disrupted. The active site of barnase in complexes is thus partly retained in the transition state of unfolding. Although the ligand could in principle perturb the unfolding pathway, there is independent evidence that indicates that similar structural changes occur upon unfolding of unligated barnase.