Enzyme Cavity Research Articles

Mapping of protein surface cavities and prediction of enzyme class by a self-organizing neural network.

An automated computer-based method for mapping of protein surface cavities was developed and applied to a set of 176 metalloproteinases containing zinc cations in their active sites. With very few exceptions, the cavity search routine detected the active site among the five largest cavities and produced reasonable active site surfaces. Cavities were described by means of solvent-accessible surface patches. For a given protein, these patches were calculated in three steps: (i) definition of cavity atoms forming surface cavities by a grid-based technique; (ii) generation of solvent accessible surfaces; (iii) assignment of an accessibility value and a generalized atom type to each surface point. Topological correlation vectors were generated from the set of surface points forming the cavities, and projected onto the plane by a self-organizing network. The resulting map of 865 enzyme cavities displays clusters of active sites that are clearly separated from the other cavities. It is demonstrated that both fully automated recognition of active sites, and prediction of enzyme class can be performed for novel protein structures at high accuracy.

Intramolecular chelate formation involving the carbonyl oxygen of acetyl phosphate or acetonylphosphonate in mixed ligand copper(II) complexes containing also 2,2′-bipyridine or 1,10-phenanthroline. A decreased solvent polarity favours the metal ion–carbonyl oxygen recognition †

The stability constants of the mixed ligand complexes formed by Cu2+, 2,2′-bipyridine or 1,10-phenanthroline (= Arm) and acetyl phosphate (AcP2−) or acetonylphosphonate (AnP2−) were determined by potentiometric pH titrations in water and in water containing 30 or 50% (v/v) 1,4-dioxane (25 °C; I = 0.1 M, NaNO3). Previous measurements with simple phosph(on)ate ligands, R-PO2−3 (R being a non-interacting residue), had established log KCu(Arm)Cu(Arm)(R-PO3)versus pKHH(R-PO3) straight-line plots and these were used now to prove that the Cu(Arm)(AcP) and Cu(Arm)(AnP) complexes possess a higher stability than is expected for a sole phosph(on)ate–Cu2+ coordination. This increased stability is attributed to the formation of six-membered chelates involving the carbonyl oxygen present in AcP2− and AnP2−. The formation degree of the six-membered chelates in the Cu(AcP), Cu(Bpy)(AcP), and Cu(Phen)(AcP) systems is very close to 75% in all three cases. For the corresponding systems with AnP2− it is shown that increasing amounts of 1,4-dioxane added to aqueous solutions favour the formation of the six-membered chelates in both the binary and the ternary complexes. It is concluded with regard to biological systems that such six-membered chelates will also be formed in mixed ligand complexes of other metal ions and that their formation degree will also be favoured by a reduced solvent polarity; both points are relevant for the situation in active-site cavities of enzymes.

Hydration energy landscape of the active site cavity in cytochrome P450cam

Hydration of protein cavities influences protein stability, dynamics, and function. Protein active sites usually contain water molecules that, upon ligand binding, are either displaced into bulk solvent or retained to mediate protein–ligand interactions. The contribution of water molecules to ligand binding must be accounted for to compute accurate values of binding affinities. This requires estimation of the extent of hydration of the binding site. However, it is often difficult to identify the water molecules involved in the binding process when ligands bind on the surface of a protein. Cytochrome P450cam is, therefore, an ideal model system because its substrate binds in a buried active site, displacing partially disordered solvent, and the protein is well characterized experimentally. We calculated the free energy differences for having five to eight water molecules in the active site cavity of the unliganded enzyme from molecular dynamics simulations by thermodynamic integration employing a three-stage perturbation scheme. The computed free energy differences between the hydration states are small (within 12 kJ mol−1) but distinct. Consistent with the crystallographic determination and studies employing hydrostatic pressure, we calculated that, although ten water molecules could in principle occupy the volume of the active site, occupation by five to six water molecules is thermodynamically most favorable. Proteins 32:381–396, 1998. © 1998 Wiley-Liss, Inc.

Hydration energy landscape of the active site cavity in cytochrome P450cam

Hydration of protein cavities influences protein stability, dynamics, and function. Protein active sites usually contain water molecules that, upon ligand binding, are either displaced into bulk solvent or retained to mediate protein-ligand interactions. The contribution of water molecules to ligand binding must be accounted for to compute accurate values of binding affinities. This requires estimation of the extent of hydration of the binding site. However, it is often difficult to identify the water molecules involved in the binding process when ligands bind on the surface of a protein. Cytochrome P450cam is, therefore, an ideal model system because its substrate binds in a buried active site, displacing partially disordered solvent, and the protein is well characterized experimentally. We calculated the free energy differences for having five to eight water molecules in the active site cavity of the unliganded enzyme from molecular dynamics simulations by thermodynamic integration employing a three-stage perturbation scheme. The computed free energy differences between the hydration states are small (within 12 kJ mol-1) but distinct. Consistent with the crystallographic determination and studies employing hydrostatic pressure, we calculated that, although ten water molecules could in principle occupy the volume of the active site, occupation by five to six water molecules is thermodynamically most favorable.

Inhibition of cruzipain visualized in a fluorescence quenched solid-phase inhibitor library assay. D-amino acid inhibitors for cruzipain, cathepsin B and cathepsin L.

A PEGA-resin was derivatized with a 3:1 mixture of hydroxymethyl benzoic acid and Fmoc-Lys(Boc)-OH and the fluorogenic substrate Ac-Y(NO2)KLRFSKQK(Abz)-PEGA was assembled on the lysine using the active ester approach. Following esterification of the hydroxymethyl benzoic acid with Fmoc-Val-OH a library XXX-k/r-XXXV containing approximately 200,000 beads was assembled by split synthesis. The resulting 'one bead, two peptides' library was subjected to extensive hydrolysis with cruzipain. One hundred darker beads were isolated and the 14 most persistently dark beads were collected and sequenced. The putative inhibitor peptides and several analogues were synthesized and found to be competitive microM to nM inhibitors of cruzipain in solution. The inhibitory activity was found to be unspecific to cruzipain when compared with cathepsins B and L and specific when compared with kallikrein. One of the inhibitors was docked into the active site of cathepsin B and was found most probably to bind to the enzyme cavity in an unusual manner, owing to the inserted D-amino acid residue.

Molecular Dynamics Simulations on the Complexes of Glucoamylase II (471) from Aspergillus awamori var. X100 with 1-Deoxynojirimycin and Lentiginosine

Molecular dynamics (MD) simulations on the complexes of glucoamylase II (471) from Aspergillus awamori var. X100 with two powerful inhibitors, 1-deoxynojirimycin and (+)-lentiginosine, have been performed, in order to build a model for these complexes in solution and to clarify the structure-activity relationship. MD calculations were carried out for 105 ps, over a 15 A sphere centered on the inhibitors. A 8 A residue-based cut-off was used, and the calculations were performed with explicit inclusion of solvent molecules. The MD structure of the complex 1-deoxynojirimycin-glucoamylase shows only minor deviations from the available X-ray structure. The MD structure of the complex of (+)-lentiginosine-glucoamylase, obtained by docking the inhibitor into the active site, suggests us a suitable orientation for the molecule into the enzyme cavity, which can rationalize the high inhibition activity found for (+)-lentiginosine towards amyloglucosidase from A. niger.

Binding of amino acid side-chains to S1 cavities of serine proteinases

The P1 or primary specificity residue of standard mechanism canonical protein inhibitors of serine proteinases, inserts into the S1 primary specificity cavity of the cognate enzyme upon enzyme-inhibitor complex formation. Both natural evolution and protein engineering often change the P1 residue to greatly alter the specificity and the binding strength. To systematize such results we have obtained all 20 coded P1 variants of one such inhibitor, turkey ovomucoid third domain, by recombinant DNA technology. The variants were extensively characterized. The association equilibrium constants were measured at pH 8.30, 21 (±2)°C, for interaction of these variants with six well characterized serine proteinases with hydrophobic S1 cavities. The enzyme names are followed by the best, worst and most specific coded residue for each. Bovine chymotrypsin Aα (Tyr, Pro, Trp), porcine pancreatic elastase (Leu/Ala, Arg, Ala), subtilisin Carlsberg (Cys, Pro, Glu), Streptomyces griseus proteinase A (Cys, Pro, Leu) and B (Cys, Pro, Lys) and human leukocyte elastase (Ile, Asp, Ile). The data set was merged with Ka values for five non-coded variants at P1 of turkey ovomucoid third domain obtained in our laboratory by enzymatic semisynthesis. The ratios of the highest to the lowest Ka for each of the six enzymes range from 106 to 108. The dominant force for binding to these pockets is the hydrophobic interaction. Excess steric bulk (too large for the pocket), awkward shape (Pro, Val and Ile), polarity (Ser) oppose interaction. Ionic charges, especially negative charges on Glu− and Asp− are strongly unfavorable. The Pearson product moment correlations for all the 15 enzyme pairs were calculated. We suggest that these may serve as a quantitative description of the specificity of the enzymes at P1. The sets of Streptomyces griseus proteinases A and B and of the two elastases are strongly positively correlated. Strikingly, chymotrypsin and pancreatic elastase are negatively correlated (−0.10). Such correlations can be usefully extended to many other enzymes and to many other binding pockets to provide a general measure of pocket binding specificity.

Thermolysin-inhibitor binding: Effect of the His231 → Ala mutation on the relative affinities of thiolate versus phosphoramidate inhibitors—a model theoretical investigation incorporating acontinuum reaction field hydration model

A series of site-directed mutagenesis experiments have been performed in our laboratory, bearing on the Zn2+ metalloprotease from Bacillus stearothermophillus, a protein very closely related to thermolysin (TLN), and their consequences on the binding of inhibitors belonging to the thiolate, carboxylate, hydroxamate, and phosphoramidate classes of compounds [A. Beaumont et al. (1995), J. Biol. Chem., Vol. 270, pp. 16803–16808]. An unexpected result related to the mutation of protonated His231, which resides at the entrance of the enzymatic cavity, into an Ala residue, shown to leave the binding affinities of thiolate inhibitors unaffected, but to abolish almost completely those of the phosphoramidate inhibitors. In order to account for such contrasting differential responses, we have undertaken a theoretical study of the comparative binding affinities of a representative thiol inhibitor, thiorphan (I) and a phosphoramidate (II), to a model of the enzymatic cavity of both the native and the mutated protein, encompassing up to 27 residues. The computations of inter- (ΔE) and intramolecular interactions were done with the sum of interactions between fragments ab initio computed procedure [N. Gresh et al. (1984), Theoret. Chim. Acta. Vol. 66, pp. 1–20; (1985), Theoret. Chim. Acta, Vol. 67, pp. 11–32; (1986), Int. J. Quant. Chem., Vol. 29, pp. 101–118; (1994), in Modelling the Hydrogen Bond, American Chemical Society Symposia, Vol. 569, D. A. Smith, Ed., American Chemical Society, pp. 82–112; N. Gresh (1995), J. Comput. Chem., Vol. 16, 856–882]. The energy balances for the His231 → Ala mutation incorporate a solvation energy contribution, computed using the Langlet et al. Continuum procedure [J. Langlet et al. (1988), J. Phys. Chem., Vol. 92, pp. 1617–1631], and this term was shown to play a decisive role in the comparative energy balances of the thiolate vs phosphoramidate inhibitors. Whereas the resolvation energy was able to compensate for the loss of ΔE induced by the H231 A mutation for the complex of I, thus accounting for its insensitivity to the mutation, such a compensation did not occur in the case of II, leading in its complexes to an overall loss of about 7 kcal/mole in the mutated cavity with respect to the native one. These results emphasized the critical role played by solvation in enzyme-inhibitor recognition processses. © 1997 John Wiley & Sons, Inc.

Three properties of relative shape envelopes of molecular electron density contours

The relative shapes of molecular electron density contour surfaces (MIDCO's), and various molecular shape constraints in solvent-solute interactions, in external electromagnetic fields and within enzyme cavities, are representable by electron densityT-hulls, introduced earlier. Three general properties ofT-hulls are proven, serving as the justification of a recently proposed computational scheme of molecular similarity measures.

TheT-hull approach to shape analysis

TheT-hull of both discrete point sets and continua is a generalization of the convex hull with respect to a reference objectT. VariousT-hulls serve as tools for shape analysis and as a basis for the introduction of a family of shape similarity measures. Some potential applications of these measures to shape problems arising in the natural sciences and a specific chemical example are discussed. The method ofT-hulls is applicable for the description of molecular shape effects in solvent-solute interactions, in external electromagnetic fields and within enzyme cavities.

Farnesyl Diphosphate Synthase Reactions of Geranyl Diphosphate Analogues Having Oxygen Atoms in Their Alkyl Chains

Abstract Seven geranyl diphosphate analogues having oxygen atoms in their alkyl chains were synthesized and examined for their reactivities as substrates in the reaction catalyzed by pig liver farnesyl diphosphate synthase. All of these compounds acted as substrates to give farnesyl diphosphate analogues. It was suggested that the enzyme cavity for the geranyl moiety is tolerant enough to accommodate alkyl moieties containing oxygen atoms.

Regulatory mechanisms in the activation of nitrogenous compounds by mammalian cytochrome P-450 isozymes.

Metabolic activation of nitrogenous compounds by the cytochrome P-450 system is a highly complex process. Inherent substrate factors, such as basicity, electronic state, lipophilicity, and conformation control binding of the diverse classes of amines to cytochrome P-450. Accommodation of these compounds in the enzyme cavity and proper orientation of the molecules are governed by intrinsic properties of the peptide structure of cytochrome P-450, which may be subject to modification by the action of effectors. On the membrane level, phospholipid might have some impact on substrate binding. On the other hand, bound amine substrate is beneficial to the productive interaction of the electron transport chains with the terminal acceptor, improving economy of the system. Certain amines appear to regulate O2 association with cytochrome P-450 and stabilize the various oxy species formed. Considering the selective prerequisites for oxidative attack by cytochrome P-450 at vulnerable nitrogen centers, many cytotoxic amines belonging to the category of relatively rigid, planar molecules undergo N-oxidative activation by the cytochrome P-450IA subfamily, while more bulky amines with flexible conformation are N-oxygenated preferentially by phenobarbital-inducible cytochromes P-450. Small differences in protein structure between the various cytochrome P-450 subforms might serve to stabilize aminium radicals to permit oxygen rebound. Collectively, the selective regulatory mechanisms operative in the bioactivation of nitrogen-containing compounds appear to be determined largely by the type of substrate used and the isozyme involved in catalysis. With respect to the latter, the interplay of the multiple cytochromes P-450 in the various organs of animal species thus serves to rationalize the differences in the particular selectivities for amine substrates. These are responsible for the mode and/or extent to which activation of nitrogenous compounds, including promutagens and procarcinogens, occurs, and this may explain the tissue-specific response to the tumorigenic action of these agents.

Streptomyces albus G serine β-lactamase. Probing of the catalytic mechanism via molecular modelling of mutant enzymes

In previous studies, several amino acids of the active site of class A beta-lactamases have been modified by site-directed mutagenesis. On the basis of the catalytic mechanism proposed for the Streptomyces albus G beta-lactamase [Lamotte-Brasseur, Dive, Dideberg, Charlier, Frère & Ghuysen (1991) Biochem. J. 279, 213-221], the influence that these mutations exert on the hydrogen-bonding network of the active site has been analysed by molecular mechanics. The results satisfactorily explain the effects of the mutations on the kinetic parameters of the enzyme's activity towards a set of substrates. The present study also shows that, upon binding a properly structured beta-lactam compound, the impaired cavity of a mutant enzyme can readopt a functional hydrogen-bonding-network configuration.

The?, ?, ?- Hull and theT-hull of a point set: Tools for the analysis of shapes and relative orientations of objects in 3d-space

Two new generalizations of the concept of convexity are introduced, suitable for detailed shape characterizations of point sets. The generalizations are also applicable for direct shape comparisons between point sets and for discrimination based on relative orientations. The methods ofα, β, γ-hull andT-hull are applicable for the description of molecular shapes in external electromagnetic fields or within enzyme cavities. These methods are suggested for the study of the similarity of shapes of interacting molecules, and for the analysis of certain biochemical interactions.

Arginine 220 is a critical residue for the catalytic mechanism of the Streptomyces albus G β-lactamase

Residue Arg220 was found to be important for the acylation of the Streptomyces albus G beta-lactamase by classical penicillins and cephalosporins bearing a carboxylate on C3 or C4. The R220L mutant exhibited strongly decreased kcat/Km values for those compounds. Conversely the acylation rates by benzylpenicillin methylester and deacetylcephalosporin C lactone were little affected, indicating a direct or indirect role of that positively charged residue in the interaction of the enzyme cavity with the negative charge of the substrate. Surprisingly that residue is not conserved in all class A beta-lactamases but when it is not present it can be seen in the known tertiary structures that the guanidinium group of another arginine side chain (Arg244) is similarly positioned. The mutation affected the behaviour of the enzyme towards cephaloridine much less than towards cephalothin. This might represent an example of substrate-assisted catalysis where the disappearance of a positive charge on the enzyme is partly compensated by the presence of a similarly charged group on one of the substrate side chains. All the experimental results are nicely explained by computer-modelling of the enzyme-substrate interactions.

Synergism between different metal ions in the dephosphorylation of adenosine 5′-triphosphate (ATP) in mixed metal ion/ATP systems, and influence of a decreasing solvent polarity (dioxane-water mixtures) on the dephosphorylation rate. Effects of Mg2+, Na+, and NH4+ ions

The initial rates of the dephosphorylation (i.e., v 0 = d[PO 4 ]/dt) of adenosine 5′-triphosphate (ATP) (=10 −3 M) in the mixed metal ion systems Cu 2+ /ATP/Mg 2+ , Ni 2+ , Zn 2+ , or Cd 2+ with the ratios 1:1:1 and 1:1:5 have been measured and compared at pH 5.5 and 50°C (I = 0.1, NaClO 4 ) with the corresponding binary ATP/M 2+ 1:2 and 1:6 systems. The remarkable result is that addition of Mg 2+ to a Cu 2+ /ATP 1:1 system accelerates the dephosphorylation rate significantly more than the same amounts of Mg 2+ accelerate the reaction in a Mg 2+ /ATP 1:1 system. The same synergism, based also on the Cu 2+ /ATP 1:1 system, is observed with Ni 2+ , but not with Zn 2+ and Cd 2+ . This observation is attributed to the formation of a prereactive state in the Cu 2+ /ATP 1:1 system, i.e., of a [Cu(ATP)] 2 4− dimer which involves purine-stacking and a Cu 2+ /N-7 interaction; the inherent reactivity in this dimer may be triggered by the addition of Mg 2+ or Ni 2+ . In Zn 2+ or Cd 2+ /ATP 1:1 systems also a prereactive state is formed and therefore no synergism is observed in a comparison with the corresponding mixed Cu 2+ systems. In agreement herewith, there is a rather pronounced synergism in the Zn 2+ /ATP Mg 2+ system at pH 7.5, and a somewhat smaller one under the same conditions in the Zn 2+ /ATP/Na + system. In the latter system the synergism may be considerably favored by reducing the solvent polarity, i.e., by changing the solvent from water to 50% (v/v) dioxane-water; similar effects, though less pronounced, are observed with Zn 2+ /UTP. In connection with the solvent effects it is recalled that the polarity in the active-site cavities of enzymes is also lower than in the bulk water. By experiments with ATP and Mg 2+ /ATP systems it is shown that Na + and NH 4 + have corresponding effects; this observation is important regarding cationic side chains of amino acid residues in proteins. Some further implications of the present results for biosystems are also indicated. The phosphate groups in TNP are labeled as α, β, and γ, where the latter refers to the terminal phosphate group (Fig. 1). If nothing else is specified, the formula PO 4 reperesents all related species which may be present in solution, i.e., H 3 PO 4 , H 2 PO 4 − , HPO 4 2− , and PO 4 3− . The term “dephosphorylation” is used for the transfer of a phosphoryl group to a water molecule; the term “hydrolysis” can also refer to this, but for the most part we use it in connection with the formation of hydroxo complexes of metal ions. The terms monomeric or dimeric complexes mean that one or two NTP 4− together with at least the corresponding equivalents of M 2+ are within the considered complex; hence, e.g., M 2 (NTP) is a monomeric (but dinuclear) nucleotide complex whereas [M(NTP)] 2 4− is a dimeric one.

Engineering a novel β-Iactamase by a single point mutation

beta-Lactamases are widespread and efficient bacterial enzymes which play a major role in bacterial resistance to penicillins and cephalosporins. In order to elucidate the role of the residues lying in a conserved loop of the enzymatic cavity of the active-site serine Streptomyces albus G beta-lactamase, modified proteins were produced by oligo-directed mutagenesis. Mutation of Asn116, which lies on one side of the active site cavity pointing to the substrate-binding site, into a serine residue resulted in spectacular modifications of the specificity profile of the enzyme. That replacement yielded an enzyme with a nearly unchanged activity towards good penicillin substrates. In sharp contrast its efficiency in hydrolysing cephalosporins was drastically reduced, the best substrates suffering the largest decrease in the second-order rate constant for serine acylation. In fact that single mutation generated a truly new enzyme behaving exclusively as a penicillinase, a situation which is never encountered to the same degree in any of the numerous naturally occurring variants of class A beta-lactamases.

Influence of Decreasing Solvent Polarity (Dioxane-Water Mixtures) on the Stability of Metal Ion Complexes Formed with Phosphate Monoesters

The acidity constants of H(R-MP)-, where R-MP2- = 4-nitrophenyl phosphate (NPhP2-), phenyl phosphate (PhP2-) and D-ribose 5′-monophosphate (RibMP2-), and the stability constants of the binary Cu(R-MP) complexes were determined by potentiometric pH titrations in aqueous solution and in 20, 30, 40 and 50% (v/v) dioxane-water mixtures. The solvent influence on the corresponding equilibrium constants is compared with the same influence on previously studied systems containing uridine 5′-triphosphate, formate or acetate. The influence of the solvent composition on the various ligand (L) systems was evaluated by constructing log KM(L) versus pKH(L) H plots; in all cases straight lines are obtained with slopes close to 1. This indicates that in all these systems, despite the different negative charges of the involved ligands, the solvent effect on proton binding and on metal ion binding is approximately of the same size: A decreasing solvent polarity resulting from the addition of increasing amounts of organic solvent to the aqueous solutions favors the affinity of the negatively charged ligands for protons and metal ions as well. Information of this type is considered important because the ‘effective’ or ‘equivalent solution’ dielectric constants in active-site cavities of enzymes are reduced compared with the dielectric constant of bulk water; i. e., in protein cavities also a decreased ‘solvent polarity’ is occurring and this is expected to affect the stability of metal ion-ligand bonds.

Enzymic Formation of β-D-Fructofuranosides from Sucrose: Activity and Selectivity of Inventase in Mixtures of Water and Alcohol

Abstract Invertase-catalysed alcoholysis of sucrose in water - primary alcohol mixtures containing up to 70% organic solvent shows 5-40% alkyl β-D-fructofuranoside formation. No formation of fructosides was observed under anhydrous conditions. The kinetics of the competing reactions of water and primary alcohol as fructosyl acceptors with sucrose as fructosyl donor were studied in order to establish the scope and limitations of the method. On a molar basis, water was a relatively unreactive acceptor. 6-Kestose formation due to sucrose itself as a fructosyl acceptor was suppressed by the presence of aliphatic alcohols. The results have been explained by assuming an nonspecific non-polar binding site to be present in the enzyme cavity near the specific polar binding site for the β-D-fructofuranosyl group of the substrate.

QUENCHING OF CHEMIEXCITED TRIPLET ACETONE BY BIOLOGICALLY IMPORTANT COMPOUNDS IN AQUEOUS MEDIUM

Abstract— Thermolysis of tetramethyl‐l,2‐dioxetane is a convenient source of triplet acetone, which can be monitored in aerated solutions by the sensitized fluorescence of 9,10‐dibromoanthracene. We have investigated the quenching of chemiexcited triplet acetone in air‐equilibrated aqueous solutions containing the 9,10‐dibromoanthracene‐2‐sulfonate ion by five classes of compounds: indoles, tyrosine derivatives, quinones, riboflavin, and xanthene dyes. Quenching rates for indoles, tyrosine and its 3,5‐dihalogenoderivatives, and xanthene dyes (kq= 108‐109M‐1 s‐1) are considerably smaller than the diffusion controlled rate, whereas those for quenchers with high electroaffinities, such as quinones (IP = 10–11 eV), approach the diffusion controlled rate (kq= 1010 M‐1 s‐1). Energy transfer for riboflavin probably occurs by a triplet‐singlet Förster type process.A comparison of the present data with previous studies of quenching of enzymically generated triplet acetone (isobutanal/O2/horseradish peroxidase) by the same classes of quenchers (except riboflavin) reveals that, independent of the nature of the quencher and the deactivation mechanism, the Stern‐Volmer quenching constants (kq t0) are systematically about one order of magnitude higher in the enzymatic system. The difference is attributed to a longer lifetime of triplet acetone in the latter case, “protected” in an enzyme cavity against collisions with dissolved oxygen.