Studies on the role of tetrazole in the activation of phosphoramidites.

The mechanism of the tetrazole-activated coupling step in the synthesis of oligonucleotides via phosphoramidites is studied with the help of model reactions: Treatment of diethoxydiisopropylaminophosphane with two equivalents of tetrazole resulted in a diethoxy-tetrazolophosphane, whose (31P)-NMR shift of 126 ppm is identical with the signal observed during internucleotide bond formation. A series of different related diethoxy-phosphorous-acid derivatives were also synthesized; their (31P)-NMR signals between 123.9 and 130.8 ppm are additional evidence for the intermediacy of a tetrazolide species. Further NMR investigations with more basic azoles showed that tetrazole is also active as a proton donor.     

Circular dichroic properties of the tyrosine residues in tetrazole analogues of opioid peptides.

CD studies on tetrazole analogues of opioid peptides show that peptides sharing the same N-terminal sequence, H-TyrPsi[CN(4)]Gly-, give very large Cotton effects of the Tyr side chain in the near-UV region. CD spectra of five such peptides: H-TyrPsi[CN(4)]Gly-Gly-Phe-Leu-OH (I), H-TyrPsi[CN(4)]Gly-Phe-Pro-Gly-Pro-Ile-NH(2) (II), H-TyrPsi[CN(4)]Gly-Phe-Pro-NH(2) (III), H-TyrPsi[CN(4)]Gly-Phe-Gly-Tyr-Pro-Ser-NH(2) (IV), and H-TyrPsi[CN(4)]Gly-Phe-Asp-Val-Val-Gly-NH(2) (V), and two others for comparison: H-Tyr-GlyPsi[CN(4)]Gly-Phe-Leu-OH (VI) and H-TyrPsi[CN(4)]Ala-Phe-Gly-Tyr-Pro-Ser-NH(2) (VII), were measured in methanol, 2,2,2-trifluoroethanol, and water at different pH values. The spectra show that the conformations of the Tyr(1) residue in peptides I-V are very similar in all solvents used but differ distinctly from those observed for VI and VII. Strong Tyr bands in the aromatic region result probably from the rigid structure of the common N-terminal part of peptides I-V. These bands are weaker for IV, which maybe due to the presence of a second Tyr residue in that peptide, giving an opposite contribution to the CD spectrum as that arising from Tyr1. It seems that the rigid structure of the N-terminal part of I-V results from the interaction of the Tyr(1) side chain and the tetrazole ring. The CD bands of the Tyr residues of VI and VII are much smaller than those of I-V in all solvents, except VII in trifluoroethanol (TFE) where Tyr bands comparable in intensity to those of I-V are observed. This spectral property may derive from the same sign contribution of both Tyr residues of VII to the CD spectrum.     

Effect of hydrophobic structure on the catalysis of nitric oxide release from zwitterionic diazeniumdiolates in surfactant and liposome media.

The effect of phospholipid liposomes and surfactant micelles on the rate of nitric oxide release from zwitterionic diazeniumdiolates, R1R2N[N(O)NO]-, with significant hydrophobic structure, has been explored. The acid-catalyzed dissociation of NO has been examined in phosphate-buffered solutions of sodium dodecylsulfate (SDS) micelles and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-[phospho-(1-glycerol)] sodium salt (DPPG) phospholipid liposomes. The reaction behavior of dibenzylamine-, monobenzylamine-, and dibutylamine-derived substrates [1]: R1 = C6H5CH2, R2 = C6H5CH2 NH2+(CH2)2, 2: R1 = C6H5CH2, R2 = NH3+(CH2)2, and 3: R1 = n-butyl, R2 = n-butyl-NH2+(CH2)6] has been compared with that of SPER/NO, 4: R1 = H2N(CH2)3, R2 = H2N(CH2) 3NH2+(CH2)4]. Catalysis of NO release is observed in both micellar and liposome media. Hydrophobic interactions contribute to micellar binding for 1-3 and appear to be the main factor facilitating catalysis by charge neutral DPPC liposomes. Binding constants for the association of 1 and 3 with SDS micelles were 3-fold larger than those previously obtained with comparable zwitterionic substrates lacking their hydrophobic structure. Anionic DPPG liposomes were much more effective in catalyzing NO release than either DPPC liposomes or SDS micelles. DPPG liposomes (at 10 mM total lipid) induced a 30-fold increase in the NO dissociation rate of SPER/NO compared to 12- and 14-fold increases in that of 1 and 3.     

Application of 2-cyanoethyl N,N,N'',N''-tetraisopropylphosphorodiamidite for in situ preparation of deoxyribonucleoside phosphoramidites and their use in polymer-supported synthesis of oligodeoxyribonucleotides.

Deoxyribonucleoside phosphoramidites are prepared in situ from 5'-O,N-protected deoxyribonucleosides and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite with tetrazole as catalyst, and the solutions applied directly on an automatic solid-phase DNA synthesizer. Using LCAA-CPG support and a cycle time of 12.5 min, oligonucleotides of 16-25 bases are obtained with a DMT-efficiency per cycle of 98.0-99.3%. The crude and fully deblocked products are of a purity comparable to that obtained using purified phosphoramidites. In case of d(G)16 the product was difficult to analyse and a better product was not obtained using doubly protected (O-6 diphenylcarbamoyl) guanine.     

Kinetic studies of reactions of iron(IV)-oxo porphyrin radical cations with organic reductants

Iron(IV)-oxo porphyrin radical cations are observed intermediates in peroxidase and catalase enzymes, where they are known as Compound I species, and the putative oxidizing species in cytochrome P450 enzymes. In this work, we report kinetic studies of reactions of iron(IV)-oxo porphyrin radical cations that can be compared to reactions of other metal-oxo species. The iron(IV)-oxo radical cations studied were those produced from 5,10,15,20-tetramesitylporphryinato-iron(III) perchlorate (1), 5,10,15,20-tetramesitylporphryinato-iron(III) chloride (2), both in CH(3)CN solvent, and that from 5,10,15,20-tetrakis(pentafluorophenyl)porphyrinato-iron(III) perchlorate (3) in CH(2)Cl(2) solvent. The substrates studied were alkenes and activated hydrocarbons diphenylmethane and ethylbenzene. For a given organic reductant, various iron(IV)-oxo porphyrin radical cations react in a relatively narrow kinetic range; typically the second-order rate constants vary by less than 1 order of magnitude for the oxidants studied here and the related oxidant 5,10,15,20-tetrakis(pentafluorophenyl)porphyrinato-iron(IV)-oxo porphyrin radical cation in CH(3)CN solvent. Charge transfer in the transition states for epoxidation reactions of substituted styrenes by oxidants 1 and 2, rho(+) values of -1.9 and -0.9, respectively, mirrors results found previously for related species. Competition kinetic reactions with a catalytic amount of porphyrin iron(III) species and a terminal oxidant give relative rate constants for oxidations of competing substrates that are somewhat smaller than the ratios of absolute rate constants. Water in CH(3)CN solutions has an apparent modest stabilizing effect on oxidant 1 as indicated in slightly reduced rate constants for oxidation reactions. The iron(IV)-oxo porphyrin radical cations are orders of magnitude less reactive than porphyrin-manganese(V)-oxo cations and a corrole-iron(V)-oxo species. The small environment effects found here suggest that high energy demanding hydrocarbon oxidation reactions catalyzed by cytochrome P450 enzymes might require highly reactive iron(V)-oxo transients as oxidants instead of the more stable, isomeric iron(IV)-oxo porphyrin radical cations.     

Impact of 1,5-disubstituted tetrazole ring on chelating ability of delta-selective opioid peptide

Complex formation between Cu(II) and three tetrazole analogues of opioid peptide-deltorphin I has been investigated. In potentiometric and spectroscopic (UV-Vis, CD and EPR) studies have been established the thermodynamic stability, speciation and structure of Cu(II) complexes with Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2 (L1), Tyr-Psi(CN4)-Gly-Phe-Asp-Val-Val-Gly-NH2 (L2), Tyr-Gly-Psi(CN4)-Phe-Asp-Val-Val-Gly-NH2 (L3) and Tyr-D-Ala-Psi(CN4)-Phe-Asp-Val-Val-Gly-NH2 (L4). The site of the insertion of tetrazole moiety Psi(CN4) into the peptide sequences has critical impact on their co-ordination ability. Comparison of the binding ability of the tetrazole analogues reveals that around physiological pH region the L3 and L4 are more effective ligands for copper(II) than L(1) and L(2). The peptide conformation changes achieved by Cu(II) co-ordination may be essential for binding of the tetrazole deltorphins at the opiate receptors.     

Effect of H2S on the formation of organic compounds from C, N,H, S model atmospheres submitted to a glow discharge.

H2S has been often invoked as the initial source of sulfur in prebiotic evolution, and several sulfur-containing compounds have been proposed as intermediates in the primordial synthesis of biologically relevant sulfur-containing chemicals. The possibilities of synthesis of the principal key intermediates by glow discharges in CH4-N2-H2S mixtures is studied. It is shown that synthesis of important intermediates such as HCN, (CN)2, CHCCN and CH3SH is possible from such mixtures if the amount of H2S is not more than 10%. For higher amounts of H2S, the syntheses are strongly inhibited.     

Kinetic data for redox reactions of cytochrome c with Fe(CN)5X complexes and the question of association prior to electron transfer

Use of rigorous equilibration kinetics to evaluate rate constants for the Fe(CN)6 4- reduction of horse-heart cytochrome c in the oxidized form, cyt c (III), has shown that limiting kinetics do not apply with concentrations of Fe(CN)6 4- (the reactant in excess) in the range 2-10 x 10(-4) M, I = 0.10 M (NaCl). The reaction conforms to a first-order rate law in each reactant, and at 25 degrees C, pH 7.2 (Tris), it is concluded that K for association prior to electron transfer is less than 200 M-1. From previous studies at 25 degrees C, ph 7.0 (10(-1) M phosphate), I = 0.242 M (NaCl), a value K = 2.4 x 10(3) M-1 has been reported. Had such a value applied, some or all of the redox inactive complexes Mo(CN)8 4-, Co(CN)6 3-, Cr(CN)6 3-, Zr(C2O4)4 4- present in amounts 5-20 x 10(-4) M would have been expected to associate at the same site and partially block the redox process. No effect on rats was observed. With the reductants Fe(CN)5(4-NH2-py)3- and Fe(CN)5(imid)3-, reactions proceeded to greater than 90% completion and rate laws were again first order in each reactant. Rate constants (M-1 sec-1) at 25 degrees C, pH 7.2 (Tris), I = 0.10 M (NaCl), are Fe(CN)6 4- (3.5 x 10(4)), Fe(CN)5(4-NH2py)3- (6.7 x 10(5), and Fe(CN)5(imid)3- (4.2 x 10(5). Related reactions in which cyt c(II) is oxidized are also first order in each reactant, Fe(CN)6 3- (9.1 x 10(6)), Fe(CN)5(NCS)3- (1.3 x 10(6)), Fe(CN)5(4-NH2py)2- (3.8 x 10(6) at pH 9.4), and Fe(CN)5(NH3)2- (2.75 x 10(6) at ph 8). Redox inactive Co(CN)6 3- (1.0 x 10(-3) M) has no effect on the reaction of Fe(CN)6 3- which suggests that a recent interpretation for the Fe(CN)6 3- oxidation of cyt c(II), I = 0.07 M, may also require reappraisal.     

Reactions of peroxyl radicals with Fe(H(2)O)(6)(2+)

The reactions of RO(2)* radicals with Fe(H(2)O)(6)(2+) were studied, R[double bond]H; CH(3); CH(2)COOH; CH(2)CN; CH(2)C(CH(3))(2)OH; CH(2)OH; CHCl(2)/CCl(3). All these processes involve the following reactions: Fe(H(2)O)(6)(2+)+RO(2)*<==>(H(2)O)(5)Fe(III)[bond]OOR(2+) K(1) approximately 250 M(-1); (H(2)O)(5)Fe(III)[bond]OOR(2+)+H(3)O(+)/H(2)O-->Fe(H(2)O)(6)(3+)+ROOH+H(2)O/OH(-); (H(2)O)(5)Fe(III)[bond]OOR(2+)+2Fe(H(2)O)(6)(2+)-->3Fe(H(2)O)(6)(3+)+ROH; 2 RO(2)*-->Products; RO(2)*+(H(2)O)(5)Fe(III)[bond]OOR(2+)-->Fe(H(2)O)(6)(2+)+products. The values of k(1) and k(3) [reaction is clearly not an elementary reaction] approach the ligand exchange rate of Fe(H(2)O)(6)(2+), i.e. these reactions follow an inner sphere mechanism and the rate determining step is the ligand exchange step. The rate of reaction is several orders of magnitude faster than that of the Fenton reaction. Surprisingly enough the K(1) values are nearly independent of the redox potential of the radical and are considerably higher than calculated from the relevant redox potentials. These results indicate that the ROO(-) ligands considerably stabilise the Fe(III) complex, this stabilisation is smaller for radicals with electron withdrawing groups which raise the redox potential of the radical but decrease the basicity of the ROO(-) ligands, two effects which seem to nearly cancel each other. Finally, the results clearly indicate that reaction (5) is relatively fast and affects the nature of the final products. The contribution of these reactions to oxidation processes involving 'Fenton-like' processes is discussed.     

Pteridine Nucleosides—New Versatile Building Blocks in Oligonucleotide Synthesis

Abstract

Chemical syntheses of 1-(2-deoxy-β-D-ribofuranosyl)lumazines and isopterins as well as 8-(2-deoxy-β-D-ribofuranosyl)-4-amino-7(8H)pteridones and -isoxanthopterins have been developed to make the structural analogs of the naturally occurring 2′-deoxyribonucleosides in the pteridine series available. The corresponding phosphoramidites have been used in machine-aided solid-support syntheses leading to new types of fluorescence labeled oligonucleotides. The effects of the various fluorophors on duplex formation and as labels for enzyme reactions is demonstrated.     

A study of the efficiency and the problem of sulfonation of several condensing reagents and their mechanisms for the chemical synthesis of deoxyoligoribonucleotides.

The efficiencies and problems of sulfonation of several condensing reagents for deoxyoligoribonucleotide synthesis have been studied. While 2,4,6-triisopropylbenzenesulfonyl chloride (TPSC1) gave very low yield and slow rate of coupling, the new 1:3 mixture of TPSC1 and tetrazole afforded excellent yield and extremely fast rate of reaction with the lowest rate of sulfonation. The difference and advantages of this mixture over triisopropylbenzenesulfonyl tetrazole (TPSTe) and mesitylenesulfonyl tetrazole (MSTe) are discussed. Mechanisms for the coupling reactions with these condensing reagents to account for the difference in their rates and yields of coupling are discussed.     

Partitioning of electrostatic and conformational contributions in the redox reactions of modified cytochromes c.

The reduction of acetylated, fully succinylated and dicarboxymethyl horse cytochromes c by the radicals CH3CH(OH), CO2.-, O2.-, and e-aq' and the oxidation of the reduced cytochrome c derivatives by Fe(CN)3-6 were studied using the pulse radiolysis technique. Many of the reactions were also examined as a function of ionic strength. By obtaining rate constants for the reactions of differently charged small molecules redox agents with the differently charged cytochrome c derivatives at both zero ionic strength and infinite ionic strength, electrostatic and conformational contributions to the electron transfer mechanism were effectively partioned from each other in some cases. In regard to cytochrome c electron transfer mechanism, the results, especially those for which conformational influences predominate, are supportive of the electron being transferred in the heme edge region.     

Can the 1,5-disubstituted tetrazole ring modify the co-ordinating ability and biological activity of opiate-like peptides?

The copper(II) complexing ability and the biological activity of beta-casomorphin-7 tetrazole analogues have been investigated. Potentiometric and spectroscopic (UV-Vis, CD and EPR) studies have been used to establish the thermodynamic stability, speciation and structure of Cu(II) complexes with YP-psi(CN4)-FPGPI-NH2 (1), YPF-psi(CN4)-AGPI-NH2 (2) and YPFP-psi(CN4)-GPI-NH2 (3). Comparison of the binding ability of the tetrazole analogues reveals that the most effective ligand for copper(II) is YPF-psi(CN4)-AGPI-NH2. The effectiveness of this ligand comes from its particular conformation suited for the Cu(II) 2N co-ordination mode in the physiological pH region. The ability of casomorphin tetrazole analogues to activate rat mast cells to histamine release in vitro in the presence of copper(II) has been studied.     

Thymidylate synthase with a C-terminal deletion catalyzes partial reactions but is unable to catalyze thymidylate formation.

The V316Am mutant of Lactobacillus casei thymidylate synthase has a single amino acid deletion at the C-terminus which abolishes catalysis of dTMP formation. However, V316Am catalyzes two partial reactions which require covalent catalysis: a CH2H4folate-dependent exchange of the 5-hydrogen of dUMP for protons in water and a thiol-dependent dehalogenation of 5-bromo- and 5-iodo-dUMP. These reactions proceed with kcat and Km values similar to those of the wild-type TS-catalyzed reactions. dUMP, dTMP, and FdUMP are competitive inhibitors of the debromination reaction with Ki values similar to those obtained with wild-type enzyme. These results show that removal of the terminal valine does not alter the ability of the enzyme to bind to or form covalent bonds with nucleotide ligands. V316Am also forms a covalent ternary complex with FdUMP and CH2H4folate. However, the affinity of the TS-FdUMP complex for the cofactor is reduced, and the rate of covalent ternary complex formation and its stability are significantly lower than with wild-type TS. These results allow us to place the major defects of the mutation on steps that occur subsequent to initial CH2H4folate binding.     

Selective O-phosphitilation with nucleoside phosphoramidite reagents.

In contrast to tetrazole, pyridine hydrochloride/imidazole converts nucleoside phosphoramidites to intermediates that show a high preference for phosphitilating hydroxyl groups relative to nucleoside amino groups. Use of this activating agent and incorporation of a pyridine hydrochloride/aniline wash step in the synthetic cycles permit synthesis of mixed base twenty-mer oligonucleotides from nucleoside reagents containing unprotected amino groups. This approach should be useful for the synthesis of oligonucleotide analogues containing substituents sensitive to reagents used in conventional deblocking steps. Pyridine hydrochloride itself is an effective reagent for activating nucleoside methylphosphonoamidites and ribonucleoside phosphoramidites, as well as deoxyribonucleoside phosphoramidites, when high O/N selectivety is not needed.     

Search for new synthetic immunosuppressants II. Tetrazole analogues of hymenistatin I

Linear and cyclic hymenistatin I (HS I) analogues with dipeptide segments Ile2-Pro3 Pro3-Pro4 and Val6-Pro7 replaced by their tetrazole analogues Ile2-psi[CN4]-Ala3', Pro3-psi[CN4]-Ala4 and Val6-psi[CN4]-Ala7 were synthesized by the solid phase peptide synthesis method and cyclized with the TBTU and/or HATU reagent. The peptides were examined for their immunosuppressive activity in the lymphocyte proliferation test (LPT).     

Effect of tetrazole moiety on coordinating efficiency of deltorphin

A study of the effect of the tetrazole moiety, a cis-amide bond surrogate, on the Cu(II) coordinating properties of oligopeptides is reported. Insertion of the tetrazole moiety Psi[CN(4)] into the peptide sequence of [D-Ala(2)]deltorphin I changes considerably the coordination ability of the peptide. Potentiometric and spectroscopic results show that if the tetrazole moiety is in a suitable position in the peptide chain, i.e. it follows the second residue, a stable CuL species involving 3N coordination is formed in the physiological pH range. The tetrazole Psi[CN(4)] ring provides one of these nitrogens. The data indicate that Cu(II) ions are strongly trapped inside a bent peptide backbone. The peptide conformation changes achieved by Cu(II) coordination may be essential for the binding of tetrazole deltorphins at opiate receptors.     

The Synthesis of RNA Containing the Modified Nucleotides N 2-Methylguanosine and N 6, N 6-Dimethyladenosine

Abstract

The phosphoramidites of the naturally occurring modified nucleotides N ²-methylguanosine and N ⁶,N ⁶-dimethyladenosine were synthesized and incorporated into short oligoribonucleotides. Described are the syntheses of the phosphoramidites and the procedures used to deprotect oligoribonucleotides in which the O ⁶ of m²G is protected with a 2-(p-nitrophenyl)ethyl group.     

Reactions of l-ergothioneine and some other aminothiones with 2,2′- and 4,4′-dipyridyl disulphides and of l-ergothioneine with iodoacetamide. 2-Mercaptoimidazoles, 2- and 4-thiopyridones, thiourea and thioacetamide as highly reactive neutral sulphur nucleophiles

1. The reactions of 2,2'- and 4,4'-dipyridyl disulphide (2-Py-S-S-2-Py and 4-Py-S-S-4-Py) with l-ergothioneine (2-mercapto-l-histidine betaine), 2-mercaptoimidazole, 1-methyl-2-mercaptoimidazole, thiourea, thioacetamide, 2-thiopyridone (Py-2-SH) and 4-thiopyridone (Py-4-SH) were investigated spectrophotometrically in the pH range approx. 1-9. 2. These reactions involve two sequential reversible thiol-disulphide interchanges. 3. The reaction of l-ergothioneine with 2-Py-S-S-2-Py and/or with the l-ergothioneine-Py-2-SH mixed disulphide, both of which provide Py-2-SH, is characterized by at least three reactive protonic states. This provides definitive evidence that neutral l-ergothioneine is a reactive nucleophile, particularly towards the highly electrophilic protonated disulphides. 4. A similar situation appears to obtain in the reactions of l-ergothioneine and Py-2-SH with 4-Py-S-S-4-Py and in the reactions of the other 2-mercaptoimidazoles, thiourea and Py-4-SH with 2-Py-S-S-2-Py. The nucleophilic reactivity of Py-4-SH suggests that general base catalysis provided by the disulphide in a cyclic or quasi-cyclic transition state is not necessary to generate nucleophilic reactivity in the other amino-thiones whose geometry could permit such catalysis. 5. The existence of a positive deuterium isotope effect in the l-ergothioneine-2-Py-S-S-2-Py system at pH6-7 provides no evidence for general base catalysis but is in accord with a mechanism involving specific acid catalysis and post-transition-state proton transfer. 6. The pH-dependences of the overall equilibrium positions of the various thiol-disulphide interchanges are described. 7. Reaction of thioacetamide with a stoicheiometric quantity of 2-Py-S-S-2-Py at pH1 provides 2 molecules of Py-2-SH per molecule of thioacetamide and elemental sulphur; these findings can be accounted for by thiol-disulphide interchange to provide a thioacetamide-Py-2-SH mixed disulphide followed by fragmentation to provide CH(3)CN, S and Py-2-SH. 8. Provision of high reactivity in the neutral forms of the members of this series of sulphur nucleophiles by electron donation by the amino group is compared with the well known alpha effect that provides enhanced nucleophilicity in compounds containing an electronegative atom adjacent to the nucleophilic atom. 9. The decrease in the u.v. absorption of l-ergothioneine at 257nm consequent on transformation of its aminothione moiety into an S-alkyl-2-mercaptoimidazole moiety provides a convenient method of following the alkylation of l-ergothioneine by iodoacetamide. 10. The pH dependence of the extinction coefficient of l-ergothioneine at 257nm is described by epsilon(257)={8x10(3)/(1+K(a)/[H(+)]} +6x10(3)m(-1).cm(-1) in which pK(a)=10.8. 11. In the pH range 3-11 the reaction is characterized by two reactive protonic states (X and XH). 12. The X state, reaction of the ionized 2-mercaptoimidazole moiety of the l-ergothioneine dianion with neutral iodoacetamide, is characterized by the second-order rate constant 4.0m(-1).s(-1) (25.0 degrees C, I=0.05). The XH state, characterized by the second-order rate constant 0.03m(-1).s(-1), is interpreted as reaction of the thione form of the neutral 2-mercaptoimidazole moiety of the l-ergothioneine monoanion with neutral iodoacetamide. 13. The XH state of the alkylation reaction does not exhibit a deuterium isotope effect.     

Kinetic parameters for the elimination reaction catalyzed by triosephosphate isomerase and an estimation of the reaction's physiological significance

Kinetic parameters for triosephosphate isomerase catalysis of the elimination reaction of an equilibrium mixture of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (DGAP) to form methylglyoxal and phosphate ion are reported for the enzyme from rabbit muscle. Pseudo-first-order rate constants for the disappearance of substrate (kelim) were determined for reactions at [Enzyme] much greater than [Substrate]. The second-order rate constant kEnz = 10.1 M-1 s-1 was determined from a plot of kelim against enzyme concentration. The kinetic parameters, determined from a steady-state kinetic analysis at [Substrate] much greater than [Enzyme], are kcat = 0.011 s-1, Km = 0.76 mM, and kcat/Km = 14 M-1 s-1. The estimated rate-constant ratio for partitioning of the enzyme-bound intermediate between protonation at carbon 2 and elimination, 1,000,000, is much larger than the ratio of 6.5 determined for the reaction of the enediolate phosphate in a loose complex with quinuclidinonium cation, a small buffer catalyst. There is a 10(5)-10(8)-fold decrease in the rate constant for the elimination reaction of the enediolate phosphate when this species binds to triosephosphate isomerase. The kinetic parameters for the elimination reaction catalyzed by the native triosephosphate isomerase and for the reaction catalyzed by a mutant form of the enzyme, which is missing a segment that forms hydrogen bonds with the phosphate group of substrate [Pompliano, D. L., Peyman, A., & Knowles, J. R. (1990) Biochemistry 29, 3186-3194] are similar.(ABSTRACT TRUNCATED AT 250 WORDS)