Unprotonated Substrate Research Articles

Reaction Mechanism of Monoamine Oxidase from QM/MM Calculations

The flavoenzyme monoamine oxidase (MAO) is essential for the enzymatic decomposition of neurotransmitters. While it is commonly accepted that the rate limiting step of the reaction is the stereoselective abstraction of a hydrogen from the substrate, the precise mechanism is unknown. We modeled the reaction of human MAO-B with benzylamine by means of QM/MM calculations based on density functional theory. Oxidation of the unprotonated substrate was found to proceed with rates in good agreement with experimental values, while the protonated substrate does not react at room temperature. Our results support a concerted asynchronous polar nucleophilic mechanism. The lone pair of the amine-nitrogen interacts with a carbon atom of the flavin cofactor. During the reaction, this lone pair, as well as a proton, are transferred to the cofactor. Analysis of the electronic structure during the reaction rules out a radical mechanism.

Isolation and Characterization of Glutaminyl Cyclases from Drosophila: Evidence for Enzyme Forms with Different Subcellular Localization

Glutaminyl cyclases (QCs) present in plants and vertebrates catalyze the formation of pyroglutamic acid (pGlu) from N-terminal glutamine. Pyroglutamyl hormones also identified in invertebrates imply the involvement of QC activity during their posttranslational maturation. Database mining led to the identification of two genes in Drosophila, which putatively encode QCs, CG32412 (DromeQC) and CG5976 (isoDromeQC). Analysis of their primary structure suggests different subcellular localizations. While DromeQC appeared to be secreted due to an N-terminal signal peptide, isoDromeQC contains either an N-terminal mitochondrial targeting or a secretion signal due to generation of different transcripts from gene CG5976. According to the prediction, homologous expression of the corresponding cDNAs in S2 cells revealed either secreted protein in the medium or intracellular QC activity. Subcellular fractionation and immunochemistry support export of isoDromeQC into the mitochondrion. For enzymatic characterization, DromeQC and isoDromeQC were expressed heterologously in Pichia pastoris and Escherichia coli, respectively. Compared to mammalian QCs, the specificity constants were about 1 order of magnitude lower for most of the analyzed substrates. The pH dependence of the specificity constant was similar for both enzymes, indicating the necessity of an unprotonated substrate amino group and two protonated groups of the enzyme, resulting in an asymmetric bell-shaped characteristic. The determination of the metal content of DromeQC revealed equimolar protein-bound zinc. These results prove conserved enzymatic mechanisms between QCs from invertebrates and mammals. Drosophila is the first organism for which isoenzymes of glutaminyl cyclase have been isolated. The identification of a mitochondrial QC points toward yet undiscovered physiological functions of these enzymes.

Kinetics and mechanism of aquation of chromium(III) complexes with 3-hydroxypicolinic and 2-hydroxynicotinic acids in HClO{4{ solutions

Two complexes, [Cr(3-hpic){3}]{0} and [Cr(2-hnic){3}]{0} (where 3-hpic = hydroxypicolinic acid and 2-hnic = 2-hydroxynicotinic acid anions), were prepared and characterized in solution. The 3-hpic ligand forms a 5-membered chelate ring via pyridine nitrogen and carboxlate oxygen atoms, whereas the 2-hnic ligand forms a 6-membered chelate ring via carboxylate and phenolate oxygen atoms. The kinetics of the acid-catalyzed aquation were studied spectrophotometrically in the 0.1–1.0 HClO{4} range, at I = 1.0 . The rate equations for the first aquation step – the chelate-ring opening – was determined and a mechanism was proposed. In the case of [Cr(3-hpic){3}]{0}, the reversible chelate-ring opening at the Cr—N bond precedes much slower than the second aquation step – a one-end bonded ligand liberation. The equation rate is of the form: k{obs} = k{1} + k{-1} /Q{1}[H{+}], where k{1} and k{-1} are the rate constants for the forward and the reverse processes in the unprotonated substrate and Q{1} is the protonation constant of the non-bonded pyridine nitrogen atom. In the case of [Cr(2-hnic)3]{0}, the chelate-ring opening at the Cr—O (phenolate) bond is the rate-determining step. The observed pseudo-first order rate constant increases as [H{+}] increases: k{obs} = k{0} + k{H}Q{H}[H{+}], where k{0} and k{H} are the rate constants of the spontaneous and acid catalyzed processes and Q{H} is the protonation constant of the coordinated phenolate oxygen atom. The results lead to the conclusion that an aquation mechanism depends on the coordination mode of the ligand.

Identification of Human Glutaminyl Cyclase as a Metalloenzyme: POTENT INHIBITION BY IMIDAZOLE DERIVATIVES AND HETEROCYCLIC CHELATORS

Human glutaminyl cyclase (QC) was identified as a metalloenzyme as suggested by the time-dependent inhibition by the heterocyclic chelators 1,10-phenanthroline and dipicolinic acid. The effect of EDTA on QC catalysis was negligible. Inactivated enzyme could be fully restored by the addition of Zn2+ in the presence of equimolar concentrations of EDTA. Little reactivation was observed with Co2+ and Mn2+. Other metal ions such as K+, Ca2+, and Ni2+ were inactive under the same conditions. Additionally, imidazole and imidazole derivatives were identified as competitive inhibitors of QC. An initial structure activity-based inhibitor screening of imidazole-derived compounds revealed potent inhibition of QC by imidazole N-1 derivatives. Subsequent data base screening led to the identification of two highly potent inhibitors, 3-[3-(1H-imidazol-1-yl)propyl]-2-thioxoimidazolidin-4-one and 1,4-bis-(imidazol-1-yl)-methyl-2,5-dimethylbenzene, which exhibited respective Ki values of 818 +/- 1 and 295 +/- 5 nm. The binding properties of the imidazole derivatives were further analyzed by the pH dependence of QC inhibition. The kinetically obtained pKa values of 6.94 +/- 0.02, 6.93 +/- 0.03, and 5.60 +/- 0.05 for imidazole, methylimidazole, and benzimidazole, respectively, match the values obtained by titrimetric pKa determination, indicating the requirement for an unprotonated nitrogen for binding to QC. Similarly, the pH dependence of the kinetic parameter Km for the QC-catalyzed conversion of H-Gln-7-ami-no-4-methylcoumarin also implies that only N-terminally unprotonated substrate molecules are bound to the active site of the enzyme, whereas turnover is not affected. The results reveal human QC as a metal-dependent transferase, suggesting that the active site-bound metal is a potential site for interaction with novel, highly potent competitive inhibitors.

Kinetics and mechanism of the first aquation stage for the [Cr(pic)3]0 and [Cr(pic)2(OH)]20 complexes in HClO4 solutions

Aquation of [Cr(pic)3]0 and [Cr(pic)2(OH)]20 in aqueous HClO4 solutions leads to formation of the common product – [Cr(pic)2(H2O)2]+. The first, reversible stage, the ring opening via Cr—N bond breaking in [Cr(pic)3]0 is followed by the second, rate-determining step – one-end bonded pic ligand liberation. In the case of the [Cr(pic)2(OH)]20 complex, the first faster stage produces the singly bridged dimer, which undergoes cleavage into the parent monomers in the second, much slower step. The subsequent aquation of [Cr(pic)2(H2O)2]+ is extremely slow and leads to [Cr(pic)(H2O)4]2+ formation, which practically does not undergo further ligand substitution under the conditions applied. Kinetics of the first aquation stage for [Cr(pic)3]0 and of the second step for [Cr(pic)2(OH)]20 were studied spectrophotometrically in the 0.1–1.0 M HClO4 range at I = 1.0 M. The observed pseudo-first order rate constant for [Cr(pic)3]0 decreases with [H+] increase according to the rate law: kobs = k1 + k−1Q1/[H+], where k1 and k−1 are the rate constants of the forward and the reverse processes in the unprotonated substrate and Q1 is the protonation constant of the pyridine nitrogen atom. In the case of the [Cr(pic)2(OH)]20 complex, the rate for the singly bridged dimer cleavage does not depend on [H+]. The activation parameters for the chelate-ring opening in [Cr(pic)3]0 and for the singly bridged dimer cleavage have been determined and discussed. Some kinetic data of the slow, second aquation stage for the [Cr(pic)3]0 complex and of the fast, first aquation stage for the doubly bridged dimer have been studied; for both reactions the rate increases linearly with the increase in [H+].

Catalysis of the beta-elimination of HF from isomeric 2-fluoroethylpyridines and 1-methyl-2-fluoroethylpyridinium salts. Proton-activating factors and methyl-activating factors as a mechanistic test to distinguish between concerted E2 and E1cb irreversible mechanisms.

Second-order rate constants, k(OH)(N), M(-)(1) s(-)(1), for the beta-elimination reactions of HF with 2-(2-fluoroethyl)pyridine (2), 3-(2-fluoroethyl)pyridine (3), and 4-(2-fluoroethyl)pyridine (4) in OH(-)/H(2)O, at 50 degrees C and mu = 1 M KCl, are = 0.646 x 10(-)(4) M(-)(1) s(-)(1), = 2.97 x 10(-)(6) M(-)(1) s(-)(1), and = 5.28 x 10(-)(4) M(-)(1) s(-)(1), respectively. When compared with the second-order rate constants for the same processes with the nitrogen-methylated substrates 1-methyl-2-(2-fluoroethyl)pyridinium iodide (5), 1-methyl-3-(2-fluoroethyl)pyridinium iodide (6), and 1-methyl-4-(2-fluoroethyl)pyridinium iodide (7), the methyl-activating factor (MethylAF) can be calculated from the ratio k(OH)(NCH)3/, and a value of 8.7 x 10(5) is obtained with substrates 5/2, a value of 1.6 x 10(3) with 6/3, and a value of 2.1 x 10(4) with 7/4. The high values of MethylAF are in agreement with an irreversible E1cb mechanism (A(N)D(E) + D(N)) for substrates 5 and 7 and with the high stability of the intermediate carbanion related to its enamine-type structure. In acetohydroxamate/acetohydroxamic acid buffers (pH 8.45-9.42) and acetate/acetic acid buffers (pH 4.13-5.13), the beta-elimination reactions of HF, with substrates 2 and 4, occur at NH(+), the substrates protonated at the nitrogen atom of the pyridine ring, even when the [NH(+)] is much lower than the [N], the unprotonated substrate, due to the high proton-activating factor (PAF) value observed: 3.6 x 10(5) for 2 and 6.5 x 10(4) for 4 with acetohydroxamate base. These high PAF values are indicative of an irreversible E1cb mechanism rather than a concerted E2 (A(N)D(E)D(N)) mechanism. Finally, the rate constant for carbanion formation from NH(+) with 2 is k(B)(NH)+ = 0.35 M(-)(1) s(-)(1), which is lower than when chlorine is the leaving group ( = 1.05 M(-)(1) s(-)(1); Alunni, S.; Busti, A. J. Chem. Soc., Perkin Trans. 2 2001, 778). This is direct experimental evidence that some lengthening of the carbon-leaving group bond can occur in the intermediate carbanion. This is a point of interest for interpreting a heavy-atom isotope effect.

Oxidation of phenethylamine derivatives by cytochrome P450 2D6: the issue of substrate protonation in binding and catalysis.

Cytochrome P450 (P450) 2D6 oxidizes a wide variety of drugs typically at a distance of 5-7 A from a basic nitrogen on the substrate. To investigate the determinants of P450 2D6 catalysis, we analyzed the binding and oxidation of phenethylamine substrates. P450 2D6 discriminated between the various phenethylamines, as evidenced by binding and steady-state results. Whereas the spectral binding affinity for 3-methoxyphenethylamine and 4-methoxyphenethylamine was similar, the affinity for 4-hydroxyphenethylamine was 12-fold weaker than for 3-hydroxyphenethylamine at pH 7.4. The binding of 3,4-dihydroxyphenethylamine was equally poor. These equilibrium dissociation constants were based on the observation of both type I and type II perturbation difference spectra; the former involves displacement of the proximal H(2)O ligand, yielding an iron spin state change, and the latter requires nitrogen ligation to the heme iron. One explanation for the observed type II binding spectra is the presence of both protonated and unprotonated forms of these compounds. To address this possibility, the K(S) values for 3-methoxyphenethylamine and 4-methoxyphenethylamine were determined as a function of pH. Two apparent pK(a) values were determined, which corresponded to a P450 2D6 residue involved in binding and to a lowered pK(a) of a substrate amine group upon binding P450 2D6. The apparent pK(a) of the enzyme residue (6.6) is much higher than the expected pK(a) of Asp301, which has been hypothesized to play a role in binding. Interestingly, the apparent pK(a) for the methoxyphenethylamine derivatives decreased by as much as 2 pH units upon binding to P450 2D6. 3-Methoxyphenethylamine and 4-methoxyphenethylamine underwent sequential oxidations with O-demethylation and subsequent ring hydroxylation to form 3,4-dihydroxyphenethylamine (dopamine). At higher substrate concentrations, the second oxidation was inhibited. This result can be explained by the increasing concentration of the inhibitory unprotonated substrate. Nevertheless, the rates of methoxyphenethylamine oxidations are the highest reported for P450 2D6 substrates.

Mechanism and proton activating factors in the base‐induced β‐elimination reaction of N‐[2‐(2‐quinolyl)ethyl]quinuclidinium salt

AbstractN‐[2‐(2‐Quinolyl)ethyl]quinuclidinium salt in OH−–H2O, 50 °C, µ = 1 M KCl undergoes an elimination reaction with formation of 2‐vinylquinoline; the second‐order rate constant is kNOH = 12.8 × 10−3 dm3 mol−1 s−1. In acetohydroxamic acid–acetohydroxamate buffers at pH 8–9 the β‐elimination reaction occurs by a reversible E1cb mechanism, (E1cb)R. In this process, carbon deprotonation occurs from the conjugate acid, protonated at the nitrogen atom of the quinoline ring, NH+; this species is present at a very low concentration with respect to the unprotonated substrate N, with pKa = 3.87 at 50 °C, µ = 1 M KCl. The reason for the high reactivity of NH+ with respect to N is related to the high stability of the intermediate carbanion formed from NH+, which presents an enamine structure. Kinetic parameters from a study of acid–base catalysis can be calculated and compared with those of the related system activated by a pyridine ring. Studies of H/D exchange and solvent isotope effect are in agreement with the proposed mechanism. Copyright © 2001 John Wiley & Sons, Ltd.

Mechanism and proton activating factors in base-induced β-elimination reactions of 2-(2-chloroethyl)pyridine †

The substrate 2-(2-chloroethyl)pyridine reacts in OH−/H2O, 50 °C, μ = 1 M KCl by an elimination reaction with the formation of 2-vinylpyridine; the second order rate constant is kNOH = 4.59 × 10−4 dm3 mol−1 s−1. In acetohydroxamate–acetohydroxamic acid buffers, the elimination reaction competes with the SN2 reaction of the acetohydroxamate nucleophile. Studies of acid–base catalysis at pH values ranging from 8.42 to 9.42 are in agreement with an E1cb irreversible mechanism, where carbon deprotonation occurs from the substrate protonated at the nitrogen of the pyridine ring (NH++++++), even if it is present at very low concentrations with respect to the unprotonated substrate (N) under the reaction conditions. The value for the reactivity ratio between NH++++++ and N is of the order of 105. The strong reactivity of NH++++++ is attributed to the high stability of the carbanion intermediate formed; this intermediate has an enamine structure.

QM/MM and SCRF studies of the ionization state of 8-methylpterin substrate bound to dihydrofolate reductase: existence of a low-barrier hydrogen bond

Using combined semiempirical quantum mechanics and molecular mechanics (QM/MM) and ab initio self-consistent reaction field (SCRF) calculations, we determined that a low-barrier hydrogen bond (LBHB) is formed when the mechanism-based substrate 8-methylpterin binds to dihydrofolate reductase (DHFR). The substrate initially was assumed bound either in the ion-pair form corresponding to N3-protonated substrate hydrogen (H) bonded to the unprotonated (carboxylate) of the conserved Glu30 residue in the active site, or in the neutral-pair form corresponding to unprotonated substrate H bonded to the neutral (carboxylic acid) form of Glu30. The free energy of interaction of these H-bonded systems with the protein/solvent surroundings was computed using a coordinate-coupled free energy perturbation (FEP) method implemented within the molecular dynamics (MD) simulation scheme and using a semiempirical (PM3) QM/MM force field. The free energy obtained from the QM/MM force-field simulations corresponds most closely with the corresponding free energy component obtained from HF/6-31G∗ SCRF calculations using a value of 2 for the dielectric constant (ϵ) for the solvated protein. Calculations were performed at levels ranging from HF/6-31G to MP2/6-31G∗ to B3LYP/6-31+G∗∗, with varying dielectric constants. The energy-minimized path for motion of the proton in the H bond along a one-dimensional reaction coordinate was calculated at HF/6-31G, HF/6-31G∗ (ϵ = 1) and B3LYP/6-31G∗ (ϵ = 2) levels. These calculations identified a second neutral-pair complex, involving the 2-amino group of substrate, which also interacts with Glu30, which is lower in energy than the ion-pair form. A harmonic vibrational analysis shows that the first vibrational state appears to lie near or above the TS connecting potential energy minima corresponding to the two neutral-pair configurations, thus indicating an LBHB. Consequently, the H-bonded system will have a significant probability of being found in the ion-pair form, in agreement with experimental spectral studies indicating an enzyme-bound cation and suggesting that the LBHB would activate substrate towards hydride-ion transfer from NADPH.

Triazene drug metabolites. Part 16.1 Kinetics and mechanism of the hydrolysis of aminoacyltriazenes

Kinetic studies of the hydrolysis of 3-aminoacyl-1-aryl-3-methyltriazenes to the corresponding 1-aryl-3-methyltriazenes were carried out in pH 3–12 aqueous buffers at 25 °C. Pseudo-first-order rate constants were found to depend both on pH and buffer concentration. pH–Rate profiles for the compounds derived from α-amino acids, 3a–e, exhibit sigmoidal curves consistent with an HO–-catalysed reaction proceeding via both protonated and unprotonated forms of the structure. For the β-amino acid derivative, 3f, the difference in reactivity between these two substrate forms is much less pronounced. For the N-acetylated derivative, 3g, only the unprotonated form is kinetically significant. Solvent deuterium isotope effects for the reaction of HO– with the protonated substrate are ca. 0.8 and for the unprotonated substrate ca. 1.0, suggesting nucleophilic catalysis by HO– for both forms. Both protonated and unprotonated forms of the substrate are subject to buffer catalysis. The Brönsted β value of 0.65 is consistent with general-base catalysis, as are the solvent deuterium isotope effect of ca. 1.3 for the imidazole-catalysed hydrolysis of the protonated substrate and the ratio of 1.1 for the piperidine- and 2,2,4,4-tetramethylpiperidine-catalysed hydrolyses of the unprotonated form.

HD exchange kinetics of alcohols and protonated peptides: Effects of structure and proton affinity

AbstractThe kinetics of gas‐phase HD exchange reactions of a series of protonated amino acids and peptides with deuterium‐substituted alcohols (D2O, CH3OD, C2H5OD and 1‐C4H9OD) were studied in an external source Fourier transform mass spectrometer. The number of exchanges observed on the time‐scale of these experiments ranged from one to the total number of ‘labile’ substrate hydrogens, depending on the amino acid and the deuterating reagent. Exchange efficiencies, k/kADO, varied from <0.001 to 0.3. Within the series ROD, the reactivity increased with increasing size of the R group. For the amino acids with alkyl side‐chains, a roughly linear correlation of log(k/kADO) with proton affinity difference (ΔPA = PA of unprotonated substrate ‐ PA of reagent) was observed. The amino acids lysine and histidine and the dipeptides alanylglycine and diglycine showed higher reactivity and greater tendency for multiple exchange, with a weaker dependence on ΔPA. The ability of a peptide and an alcohol to exchange efficiently even when ΔPA is larger is attributed to the occurrence of exchange within a cyclic hydrogen‐bonded complex, in which the deuterating agent forms a bridge between the site of protonation and a basic site on the substrate.

Aromatic sulphonation. Part 92. Sulphonation of the three methylphenols and the six dimethylphenols in concentrated aqueous sulphuric acid; and the lsomerization of some of the resulting sulphonic acids and of m-xylene-2-and o-xylene-3-sulphonic acid

The isomer distributions for the mono- and di-sulphonation of the three methylphenols in 81.6–90.0% sulphuric acid and of the six dimethylphenols in 84.9% sulphuric acid at 35 °C have been determined by means of 1H n.m.r. spectroscopy. Products that would result from demethylation, disproportionation, or ipso-substitution have not been observed. The monosulphonation isomer distribution of each of the three methylphenols is independent of the sulphuric acid concentration. 2,4-Dimethylphenol in 84.9% H2SO4yields 19% 5- and 79% 6-sulphonic acid, and 2,6-dimethylphenol yields 28% 3- and 72% 4-sulphonic acid. With the phenols (2), (3), (5), and (7), which have positions both ortho and para to the –OH substituent available for sulphodeprotonation, the yield of ortho-substitution is relatively high, viz. 44, 45,36, and 27%, respectively. The mono- and di-sulphonic acids of which the sulpho group is between the –OH and an –Me group are unstable under the reaction conditions employed, with the exception of the 3,5-dimethylphenol-2-sulphonic acid. It is proposed that their isomerization proceeds by protiodesulphonation followed by resulphonation to yield the more stable sulphonic acid isomer(s). From a kinetic analysis of the calculated first-order rate coefficients for protiodesulphonation, it is concluded that the unprotonated substrate species is the entity undergoing the protiodesulphonation.

Aromatic sulphonation. Part 91. The sulphonation of anisole, phenol, phenyl methanesulphonate, potassium phenyl sulphate, and a series of methyl-, bromo-, and chloro-substituted anisoles and phenols in concentrated aqueous sulphuric acid

The (homogeneous) sulphonation of a number of aromatic ethers and alcohols, viz. anisole (1), 3- methyl-(2), 4-methyl-(3). 2-bromo-(4), 4-bromo-(5), 2-chloro-(6), and 4-chloro-anisole (7), phenol (8), 2-methyl-(9), 3-methyl-(10), 4-methyl-(11), 4-bromo-(12), 2-chloro-(13), and 4- chloro-phenol (14), phenyl methanesulphonate (15), and potassium phenyl sulphate (16) in concentrated aqueous sulphuric acid at 25.0 °C has been studied, and rates and isomer distributions have been determined. The sulphonation is first-order in the aromatic substrate, and from the rate measurements it is concluded that the species undergoing sulphonation in the phenyl ring is an unprotonated substrate species. In the lower acid concentrations the sulphonating entity is H3SO4+. With increasing sulphuric acid concentration there is a gradual change-over in the sulphonating entity from H3SO4+ to H2S2O7. The acid concentrations of equal rate contribution by the two entities for anisole and phenol are 87 and 90 ± 1%, respectively. Sulphonation on the oxygen atom (i.e., sulphation) does not occur. The o/p-ratios for (1) and (8) do not vary over the studied sulphuric acid range of 75–90% H2SO4. Partial rate factors for the 2- and 4-position of (1) and (8) are reported. The very low partial rate factors for the 4-substitution of (1) and (8) and the observed extreme suppression and compression of the reactivities of the substrates (1)–(14) are ascribed to hydrogen bonding of the substrates with the acidic solvent species present. It is tentatively suggested that the relatively high contents of sulphonation ortho to –OR with anisole (36%) and phenol (48%) are due to specific complexation of the substrates with the sulphonating electrophile.

The α-Chymotrypsin-catalyzed Hydrolysis of Esters of α-Amino Acids

Abstract The pH dependence of the α-chymotrypsin-catalyzed hydrolysis of l-tryptophan methyl ester in H2O and D2O and of d-tryptophan methyl ester was determined by spectroscopic means (kcat, Km and kcat/Km). No product inhibition or substrate activation was observed. The kinetic parameters of the α-chymotrypsin-catalyzed hydrolysis of l-tyrosine p-nitrophenyl ester were also determined. Values of kcat for both the tyrosine and tryptophan derivatives depend on the ionization of the amino group. For the tryptophan derivatives which were investigated in most detail, the pH dependence of kcat/Km ( = k2/Ks) yields a bell-shaped curve with a maximum at pH 7.2. The deuterium oxide kinetic solvent isotope effect and the dependency of the reaction on a base of pK ∼ 7 for the l derivative are comparable to that seen with the corresponding acylamino derivative. Thus, the mechanism of hydrolysis of the amino acid esters appear to be the same as that for the acylamino acid esters. However, the ionizability of the α-amino group in the former produces a markedly different pH dependency of the enzyme-catalyzed reaction, which can be attributed to the presence of a protonated and unprotonated substrate. The protonated substrate is about 20-fold faster than the unprotonated substrate in both acylation and deacylation of l-tryptophan methyl ester. Full specificity of the enzyme (l over d) is found for l-tryptophan methyl ester in its protonated form. This may be due to a hydrogen bond between the substrate and the enzyme with very strict orientational requirements.