The palladium(II) promoted hydrolysis of the methyl esters of glycyl-L-leucine,glycyl-L-alanine and L-alanylglycine

The palladium(II)-promoted hydrolysis of the methyl esters of glycyl-L-leucine, glycyl-L-alanine and L-alanylglycine have been studied at 25 °C and I=0.1 M in the pH range 4–5. At a 1:1 metal to ligand ratio the peptide esters act as tridentate ligands, donation occurring via the terminal amino group, the deprotonated amide nitrogen, and the carbonyl group of the ester. Due to the high Lewis acidity of Pd(II) rapid hydrolysis of the ester function by water and hydroxide ion occurs. Rate constants k_OH and k_H2O have been obtained for base hydrolysis and water hydrolysis of the coordinated peptide esters at 25 °C. The rate constants for base hydrolysis are 3.4 X 10⁶ M⁻¹ s⁻¹ (L-alaglyOMe), 6.4 X 10⁶ M⁻¹ s⁻¹ (gly-L-alaOMe) and 2.3 X 10⁷ M⁻¹ s⁻¹ (gly-L-leuOMe). Base hydrolysis of the coordinated peptide esters is at least 10⁶ times that of the free unprotonated ligand. Activation parameters have been obtained for both water and base hydrolysis of the Pd(II) complex of methyl L-alanylglycinate and possible mechanisms for the hydrolyses are considered.     

Binding and hydrolysis studies of antitumoural titanocene dichloride and Titanocene Y with phosphate diesters

The interaction of the antitumoural metallocene dihalides, titanocene dichloride (Cp₂TiCl₂) and Titanocene Y (bis-[(p-methoxybenzyl)cyclopentadienyl]titanium(IV) chloride), with bis(4-nitrophenyl) phosphate (BNPP), which is a widely used model for the phosphate diester linkages in DNA, has been studied. Cp₂TiCl₂ has been shown to promote the cleavage of the phosphate diester in weakly acidic solution. At pH 4, 37 °C, a 10⁶-fold rate acceleration over the uncatalysed reaction was observed under pseudo-first-order conditions, when freshly prepared solutions of Cp₂TiCl₂ were applied. The activity of aged solutions dropped significantly due to the formation of insoluble precipitates of hydrolysed Ti species. The precipitates isolated from aged solutions were shown to act as moderately active, heterogeneous catalysts for BNPP cleavage. By contrast, no hydrolysis of the phosphate diester could be observed in the presence of Titanocene Y. Implications for the mode of action of the apoptosis-inducing metallocene dihalides are discussed.     

Metallo-β-lactamase and phosphotriesterase activities of some zinc(II) complexes

Metallo-β-lactamases (mβl) and phosphotriesterase (PTE) are zinc(II) enzymes, which hydrolyze the β-lactam antibiotics and toxic organophosphotriesters, respectively. In the present work, we have synthesized a few asymmetric phenolate-based ligands by sequential Mannich reaction and their corresponding zinc(II) complexes. These zinc(II) complexes were studied for their mβl and PTE activities. It is shown that the zinc(II) complexes can hydrolyze oxacillin, the β-lactam antibiotic, at much higher rates as compared to the hydrolysis of p-nitrophenyl diphenylphosphate (PNPDPP), the phosphotriester. Among the complexes studied, the binuclear asymmetric complex 1 having a water molecule coordinated to one of the zinc(II) ions exhibits much better mβl activity than the mononuclear complexes. However, the mononuclear zinc(II) complexes having labile chloride ions exhibit significant PTE activity, which can be ascribed to the replacement of chloride ions by hydroxide ions during hydrolysis reactions.     

Phosphodiesterolytic activity of lanthanide (III) complexes with α-amino acids

Kinetics of the hydrolysis of BNPP (bis(4-nitrophenyl)phosphate) mediated by lanthanide - samarium (III) and ytterbium (III) - alone and in the presence of various alfa amino acids has been systematically studied at 37.0 °C and I = 0.15 M in NaClO₄, in the pH interval of 7-9. The rate of BNPP cleavage is sensitive to metal ion concentration, pH, and ligand to metal molar ratio. Hydrolysis follows Michaelis-Menten-type saturation kinetics. For both metals, high pH values markedly increase the observed activity. Besides, potentiometric titrations of all these systems under identical conditions allowed us to identify the active coordination compounds towards hydrolysis. The results show that complexes with phosphodiesterolytic activity are monomeric cationic species such as [Ln(aa)₃(OH)]²⁺ or [Ln(aa)₂(OH)₂]⁺. Since phosphodiesterolytic activity is evident above pH 7 and it is increased with increasing pH, hydrolytic reactions of the metals are competitive processes that could lead to their precipitation as Ln(OH)₃(s). In this sense, ligand excess (for example, ligand to metal molar ratio equal to 30) was employed. Furthermore, due to its more extended hydrolysis, ytterbium shows, in general, less activity than samarium under the studied conditions. In general, a good phosphodiesterolytic activity is observed for these complexes under similar conditions to the physiological ones. Amino acids could be easily derivatized without changing their coordinating ability, leading to lanthanide complexes possibly capable of efficiently hydrolyzing the phosphodiester linkages of nucleic acids.     

Synthesis and X-ray structure of the dinuclear platinum(II) complex with saccharin {K[Pt(sac)3(H2O)] · H2O}2: Studies on its antiproliferative activity in aqueous solution

Synthesis and X-ray structure of a dinuclear platinum(II) complex with the ligand saccharin(sac) are described. The structure shows two approximately square-planar platinum centers. Each platinum atom is coordinated to one water molecule and three N-bonded saccharinate ligands. The two centers are linked through two potassium atoms. Each potassium atom interacts with six oxygen atoms from hydration and coordinated water molecules and from carbonyl and sulfonate groups of the ligands. It is suggested that, in aqueous solution, the dimeric structure of the complex is dissociated and the monomeric species K[Pt(sac)₃(H₂O)] is formed. The complex was dissolved in water and submitted to in vitro cytotoxic analyses using HeLa cells (human cervix cancer). It was shown that the monomeric complex elicited a potent cytotoxic activity when compared to the vehicle-treated cells. The IC₅₀ value for the monomeric complex is 6.8 μM, a little bit higher than that obtained for cisplatin.     

Phosphodiester hydrolysis and specific DNA binding and cleavage promoted by guanidinium-functionalized zinc complexes

Two new Zn(II) complexes containing guanidinium groups, [Zn(L¹)Cl₂](ClO₄)₂ · H₂O · CH₃OH (1) and [Zn(L²)Cl₂](ClO₄)₂ · 0.5H₂O (2), were synthesized and characterized (L¹ = 5,5′-di[1-(guanidyl)methyl]-2,2′-bipyridyl bication and L² = 6,6′-di[1-(guanidyl)methyl]-2,2′-bipyridyl bication). Both complexes are able to catalyze bis(p-nitrophenyl) phosphate (BNPP) hydrolysis efficiently. Obtained kinetic data reveal that both 1 and 2 show nearly 300- and 600-fold rate enhancement of BNPP hydrolysis, respectively, compared to their simple analogue without the guanidinium groups [Zn(bpy)Cl₂] (bpy = 2,2′-bipyridy) (3). Enhanced acceleration for cleavage of BNPP could be attributed to cooperative interaction between the Zn(II) ion and the guanidinium groups by electrostatic interaction and H-bonding. Studies on inhibition of sequence-specific endonucleases (DraI and SmaI) by complexes show that 1 and 2 are able to recognize nucleotide sequence, -TTT^AAA-, and highly effectively cleave the plasmid DNA in the presence of hydrogen peroxide, while 3 has no specific binding to the DNA target sequences and only shows low DNA cleavage activity.     

The uncatalysed and the copper(II) promoted hydrolysis of 4-nitrophenyl L-leucinate

The uncatalysed hydrolysis of 4-nitrophenyl L-leucinate has been studied in detail over a range of pH and temperature at I=0.1 M (KNO₃). Base hydrolysis of the ester is strongly promoted by copper(II) ions. Rate constants have been obtained for the following reactions (where EH⁺ is the N- protonated ester and E is the free base form) EH⁺ + OH⁻ → products E + OH⁻ → products E + H₂O → products CuE²⁺ + OH⁻ → products Base hydrolysis of the copper(II) complex CuE²⁺ is 3.8 × 10⁵ times faster than that of E and 75 times faster than that of EH⁺ at 25 °C and I=0.1 M. Activation parameters for these reactions have been determined and possible mechanisms are considered.     

Reaction pathways for Zn(II)-catalyzed carboxylic acid esters hydrolysis

Reaction pathways have been investigated by quantum-mechanical procedures on gas phase models for the hydrolysis of methyl acetate catalyzed by a monohydroxo-Zn(II) complex formed by a phenanthroline-containing polyamine macrocycle. Based on consideration of energy barrier heights, the hydrolysis process is predicted to be bimolecular, consistently with kinetic data obtained for the hydrolysis of p-nitrophenyl acetate promoted by the modelled catalyst. Differences with respect to results of theoretical studies on the more extensively investigated hydrolysis processes catalyzed by the OH⁻ anion are discussed and appear to be mostly due to the presence of the metal centre close to the OH function in the system now investigated. The pervasive presence and path-controlling role of hydrogen bonds are also discussed.     

Preparation, crystal structures and spectroscopic properties of novel [MCl3 − n(P)3 + n] (M = Co, Rh; n = 0, 1, 2 or 3) series of complexes containing tripodal tridentate phosphine, 1,1,1-tris(dimethylphosphinomethyl)ethane

Cobalt(III) and rhodium(III) complexes of the series of [M^IIICl_3 − n(P)_3 + n]ⁿ⁺ (M = Co or Rh; n = 0, 1, 2 or 3) have been prepared with the use of 1,1,1-tris(dimethylphosphinomethyl)ethane (tdmme) and mono- or didentate phosphines. The single-crystal X-ray analyses of both series of complexes revealed that the M-P and M-Cl bond lengths were dependent primarily on the strong trans influence of the phosphines, and secondarily on the steric congestion around the metal center resulting from the coordination of several phosphine groups. In fact, the M-P(tdmme) bonds became longer in the order of [MCl₃(tdmme)] < [MCl₂(tdmme)(PMe₃)]⁺ < [MCl(tdmme)(dmpe)]²⁺ (dmpe = 1,2-bis(dimethylphosphino)ethane) < [M(tdmme)₂]³⁺ for both Co^III and Rh^III series of complexes, while the M-Cl bond lengths were shortened in this order (except for [M(tdmme)₂]³⁺). Such a steric congestion around the metal center can also account for the structural and spectroscopic characteristics of the series of complexes, [MCl(tdmme)(dmpm, dmpe or dmpp)]²⁺ (dmpm = bis(dimethylphosphino)methane, dmpp = 1,3-bis(dimethylphosphino)propane). The X-ray analysis for [CoCl(tdmme)(dmpm or dmpe)](BF₄)₂ showed that all Co-P bonds in the dmpm complex were shorter by 0.03-0.04 Å than those in the dmpe complex. Furthermore, the first d-d transition energy of the Co^III complexes and the ¹J_Rh-P(tdmme) coupling constants observed for the Rh^III complexes indicated an unusual order in the coordination bond strengths of the didentate diphosphines, i.e., dmpm > dmpe > dmpp.     

Multifunctional catalysis by metal complexes: 1. Ester hydrolysis catalyzed by oximinatozinc(II) ions

Hydrolysis of p-nitrophenyl acetate catalyzed by a Zn(II) complex of 2-acetylpyridineketoxime or 2-pyridinecarboxaldoxime was studied as a model of multifunctional catalysis by metalloproteases. The reaction proceeded exclusively through the formation of an acylcatalyst intermediate under the experimental conditions, and both the formation and the breakdown of the acyl intermediate were much faster than the spontaneous reaction. The metal ion, the metal-bound water molecule or hydroxide ion, the oximate ion, and general bases contributed to the multifunctional catalysis in ester hydrolysis by the oximinatozinc(II) ions.     

Equilibrium and kinetics of the dinuclear complex formation between N,N′-ethylenebis(salicylideneiminato)copper(II) and metal(II,I) ions in acetonitrile

The equilibrium constants and rate constants of the reactions between N,N′-ethylenebis(salicylideneiminato)copper(II) ([Cu(salen)]) and metal(II,I) ions in acetonitrile have been spectrophotometrically determined. [Cu(salen)] acts as a didentate ligand to form a dinuclear complex. The rate constants for the very labile Mn(II), Fe(II), and Zn(II) ions were directly evaluated using a variable flow-rate instrument that was newly constructed for this study. The rate constants of the dinuclear complex formation for a series of metal(II) ions vary in parallel with those of the acetonitrile solvent exchange on the corresponding metal(II) ions. This finding indicates that the dinuclear complex formation reaction of the metal(II) ions proceeds via almost the same reaction mechanism as for the acetonitrile solvent exchange reaction.     

X-ray structure and spectroscopic characterization of divalent dinuclear cobalt complexes containing carboxylate- and phosphodiester- auxiliary bridges

Two dinuclear spin-coupled divalent cobalt complexes, [Co₂(P1-O)(μ₂-OAc)](ClO₄)₂, (1) and [Co₂(P1-O)(μ₂-BNPP)](ClO₄)₂, (2) containing μ-1,3 acetate (OAc) and bis(4-nitrophenyl)phosphate (BNPP) auxiliary bridges, respectively, were synthesized by the reaction of a classic dinucleating ligand, P1-OH with cobalt(II) perchlorate in presence of acetic acid/bis(4-nitrophenyl)phosphate. They were characterized by single crystal X-ray diffraction, to show a trigonal bipyramidal geometry around each cobalt center and the intervening bridging atoms that are responsible for spin-transfer between the two divalent cobalt centers; the alkoxo oxygen donor occupies an equatorial position, and the auxiliary ligand oxygens (OAc/BNPP) occupy the axial positions. Solution state magnetic moment measurement together with UV-Vis/NIR spectra revealed a high-spin ground state (S = 3/2) for Co(II) in these compounds. Complexes 1 and 2 show interesting ¹H NMR spectral features of resonances with relatively narrower linewidths in conjunction with a sizable chemical shift dispersion of −5 and 265 ppm. Complex 2 containing the bis(4-nitrophenyl)phosphate auxiliary bridge showed narrower spectral window than complex 1 that has the acetate auxiliary bridge.     

A Mn(II)–Mn(II) center in human prolidase

Human prolidase, the enzyme responsible for the hydrolysis of the Xaa-Pro/Hyp peptide bonds, is a key player in the recycling of imino acids during the final stage of protein catabolism and extracellular matrix remodeling. Its metal active site composition corresponding to the maximal catalytic activity is still unknown, although prolidase function is of increasing interest due to the link with carcinogenesis and mutations in prolidase gene cause a severe connective tissue disorder. Here, using EPR and ICP-MS on human recombinant prolidase produced in Escherichia coli (hRecProl), the Mn(II) ion organized in a dinuclear Mn(II)–Mn(II) center was identified as the protein cofactor. Furthermore, thermal denaturation, CD/fluorescence spectroscopy and limited proteolysis revealed that the Mn(II) is required for the proper protein folding and that a protein conformational modification is needed in the transition from apo- to Mn(II)loaded-enzyme. The collected data provided a better knowledge of the human holo-prolidase and, although limited to the recombinant enzyme, the exact identity and organization of the metal cofactor as well as the conformational change required for activity were proven.     

Phosphodiesterolytic activity of samarium(III) mixed ligand complexes containing crown ethers and α-amino acids

Phosphodiesterolytic activity of samarium complexes containing crown ethers and amino acids was systematically studied. Formation constants of mixed ligand Sm–crown ethers–amino acids complexes (crown ethers = 18-crown-6, 15-crown-5 and 12-crown-4 and amino acids = Gly and Arg) were determined at 37.0 °C and 0.50 M NMe₄Cl. Kinetics of the hydrolysis of BNPP (bis(4-nitrophenyl)phosphate) mediated by lanthanide(III)-mixed ligands complexes was studied under the same experimental conditions. The rate of BNPP cleavage is sensitive to metal ion concentration, pH, and ligand to metal molar ratio. Hydrolysis follows Michaelis–Menten-type saturation kinetics. High pH values markedly increase the observed activity. Potentiometric titrations results together with kinetic data of all these systems, under identical conditions, allowed us to identify the active species towards hydrolysis. Complexes with phosphodiesterolytic activity are monomeric hydroxylated cationic species. In general, a good phosphodiesterolytic activity is observed for these complexes under similar conditions to the physiological ones.     

Structure of the bacteriophage lambda Ser/Thr protein phosphatase with sulfate ion bound in two coordination modes

The protein phosphatase encoded by bacteriophage lambda (lambda PP) belongs to a family of Ser/Thr phosphatases (Ser/Thr PPases) that includes the eukaryotic protein phosphatases 1 (PP1), 2A (PP2A), and 2B (calcineurin). These Ser/Thr PPases and the related purple acid phosphatases (PAPs) contain a conserved phosphoesterase sequence motif that binds a dinuclear metal center. The mechanisms of phosphoester hydrolysis by these enzymes are beginning to be unraveled. To utilize lambda PP more effectively as a model for probing the catalytic mechanism of the Ser/Thr PPases, we have determined its crystal structure to 2.15 A resolution. The overall fold resembles that of PP1 and calcineurin, including a conserved beta alpha beta alpha beta structure that comprises the phosphoesterase motif. Substrates and inhibitors probably bind in a narrow surface groove that houses the active site dinuclear Mn(II) center. The arrangement of metal ligands is similar to that in PP1, calcineurin, and PAP, and a bound sulfate ion is present in two novel coordination modes. In two of the three molecules in the crystallographic asymmetric unit, sulfate is coordinated to Mn2 in a monodentate, terminal fashion, and the two Mn(II) ions are bridged by a solvent molecule. Two additional solvent molecules are coordinated to Mn1. In the third molecule, the sulfate ion is triply coordinated to the metal center with one oxygen coordinated to both Mn(II) ions, one oxygen coordinated to Mn1, and one oxygen coordinated to Mn2. The sulfate in this coordination mode displaces the bridging ligand and one of the terminal solvent ligands. In both sulfate coordination modes, the sulfate ion is stabilized by hydrogen bonding interactions with conserved arginine residues, Arg 53 and Arg 162. The two different active site structures provide models for intermediates in phosphoester hydrolysis and suggest specific mechanistic roles for conserved residues.     

Enantiomer-selective solvolysis catalysed by a histidine-containing cyclic dipeptide carrying chiral side chain

Tripeptides with cyclic dipeptide backbones, cyclo[l-Glu(l-Leu-O Bzl)-t-His] and cyclo[l-Glu(l-Leu-OH)-Ir His, and the corresponding tripeptides with linear backbones, Me₃COCO-l-Glu(l-Leu-OBzl)-l-His-OMe and Me₃COCO-l-Glu(l-Leu-OH)-l-His-OMe, were synthesized and used as catalysts for the hydrolysis of carboxylic acid active esters of various types. The experimental results are summarized as follows. (I) In the hydrolysis of a neutral and hydrophobic substate, p-nitrophenyl laurate, in 20% dioxane/H₂O mixture of pH 7.8, a hydrophobic and flexible peptide, Me₃COCO-l-Glu(l-Leu-OH)-l-His-OMe, was more reactive than imidazole. On the other hand, cyclo[l-Glu(l-Leu-OBzl)-l-His] and cyclo[l-Leu-OH)-l-His], which have rigid backbone chain and fixed sidechain conformation, were not particularly reactive. (2) in the solcolysis of a positively charged substrate, p-nitrophenyl glycinate hydrochloride, in 42% i-PrOH/H₂O mixture at pH 6.95, a positively charged substrate, p-nitrophenyl glycinate hydrochloride, in 42% i-PrOH/H₂O mixture at pH 6.95, a negatively charged and flexible peptide, Me₃COCO-l-Glu(l-Leu-OH)-l-His-OMe, was more reactive than imidazole. However, cyclo [l-Glu(l-Leu-OH)-l-His] was not particularly reactive in the same reaction. In the hydrolysis of p-nitrophenyl glycinate hydrochloride in aqueous solution at pH 7.8 a hydrophobic and rigid peptide, cyclo[(l-Glu(l-Leu-OBzl)-l-His], was more reactive than imidazole. However, in the hydrolysis of p-nitrophenyl CO-AMINODODECANOATE hydrochloride, which has a positive charge and a rective site separated by a long hydrophobic chain, peptide catalysts did not show efficient catalysis. (3) In the hydrolysis of a positively charged, hydrophobic and chiral substrate, p-nitrophenyl leucinate hydrochloride, in aqueous solution at pH 6.95, the d-enantiomer was hydrolysed more quickly that the t-enantiomer with cyclo[l-Glu(l-Leu-OBzl)l-His] or cyclo[t-Glu(l-Leu-OH)-l-His] as catalyst. On the other hand, the tripeptides with linear backbone did not effect an enantiomer-selective catalysis. The solvolytic reaction catalysed by the tripeptides with cyclic dipeptide backbone in 42% i-PrOH/water mixture was also enantiomer-selective.     

Copper(II), nickel(II) and zinc(II) complexes of N-acetyl-His-Pro-His-His-NH2: equilibria, solution structure and enzyme mimicking

The copper(II), nickel(II) and zinc(II) binding ability of the multi-histidine peptide N-acetyl-His-Pro-His-His-NH₂ has been studied by combined pH-potentiometry and visible, CD and EPR spectroscopies. The internal proline residue, preventing the metal ion induced successive amide deprotonations, resulted in the shift of this process toward higher pH values as compared to other peptides. The metal ions in the parent [ML]²⁺ complexes are exclusively bound by the three imidazole side chains. In [CuH₋₁L]⁺, formed between pH 6-8, the side chains of the two adjacent histidines and the peptide nitrogen between them are involved in metal ion binding. The next deprotonation results in the proton loss of the coordinated water molecule (CuH₋₁L(OH)). The latter two species exert polyfunctional catalytic activity, since they possess superoxide dismutase-, catecholase- (the oxidation of 3,5-di-tert-butylcatechol) and phosphatase-like (transesterification of the activated phosphoester 2-hydroxypropyl-4-nitrophenyl phosphate) properties. On further increase of the pH rearrangement of the coordination sphere takes place leading to the [CuH₋₃L]⁻ species, the deprotonated amide nitrogen displaces a coordinated imidazole nitrogen from the equatorial position of the metal ion. The shapes of the visible and CD spectra reflect a distorted arrangement of the donor atoms around the metal ion. In presence of zinc(II) the species [ZnL]²⁺ forms only above pH 6, which is shortly followed by precipitation. On the other hand, the [NiL]²⁺ complex is stable over a wide pH range, its deprotonation takes place only above pH 8. At pH 10 an octahedral NiH₋₂L species is present at first, which transforms slowly to a yellow square planar complex.     

Dinuclear zinc(II) complex with novel tripodal polyamine ligand: synthesis, structure and kinetic study of carboxy ester hydrolysis

An imidazole-containing tripodal polyamine ligand N(1)-(2-aminoethyl)-N(1)-(2-imidazol-1-ylethyl)-ethane-1,2-diamine (L) was prepared and its dinuclear zinc(II) complex [Zn(L)(H(2)O)](2)(ClO(4))(4).4H(2)O (1) was obtained and examined as a catalyst for the hydrolysis of 4-nitrophenyl acetate (NA). X-ray crystal structure analysis of the complex revealed that the complex features a dinuclear cation unit with a Zn...Zn distance of 8.34A and both Zn(II) centers adopt distorted trigonal-bipyramid geometry. The solution complexation investigation performed at 25 degrees C by means of potentiometric titration revealed that the mononuclear species [ZnL](2+) is predominating in the pH rage of 7.0-9.7 in the solution and the pK(a1) for the Zn-bound water is 8.50+/-0.01. Complex 1 promoted hydrolysis of NA showed a second-order rate constant of 0.046+/-0.004 M(-1)s(-1) at pH 9.0 in 10% (v/v) CH(3)CN aqueous solution at 25 degrees C. The pH-rate profile for the second-order rate constant of NA hydrolysis with complex 1 gave a sigmoidal curve. And the results show that in the hydrolysis process the two Zn(II) centers of the dinuclear deprotonated species do not cooperate with each other and the Zn-bound hydroxide servers as reactive nucleophile toward the ester.     

Characterization of a new cutinase from Thermobifida alba for PET-surface hydrolysis

A new cutinase from Thermobifida alba (Tha_Cut1) was cloned and characterized for polyethylene terephthalate (PET) hydrolysis. Tha_Cut1 showed a high degree of identity to a T. cellulolysitica cutinase with only four amino acid differences outside the active site area, according to modeling data. Yet, Tha_Cut1 was more active in terms of PET surface hydrolysis leading to considerable improvement in hydrophilicity quantified based on a decrease of the water contact angle from 87.7° to 45.0°. The introduction of new carboxyl groups was confirmed and measured after esterification with the fluorescent reagent alkyl bromide, 2-(bromomethyl) naphthalene (BrNP), resulting in a fluorescence emission intensity increase from 980 to 1420 a.u. On the soluble model substrates p-nitrophenyl acetate (PNPA) and p-nitrophenyl butyrate (PNPB), the cutinase showed Km values of 213 and 1933 μM and k_cat values of 2.72 and 6.03 s^?1 respectively. The substrate specificity was investigated with bis(benzoyloxyethyl)terephthalate (3PET) and Tha_Cut1 was shown to release primarily 2-hydroxyethyl benzoate. This contrasts with the well-studied Humicula insolens cutinase which preferentially liberates terminal benzoic acid from 3PET.