Lawrence R. Gahan

Reactions of cisplatin hydrolytes with methionine, cysteine, and plasma ultrafiltrate studied by a combination of HPLC and NMR techniques.

The reactions of cis-[PtCl(NH3)2(H2O)]+ with L-methionine have been studied by 1D 195Pt and 15N NMR, and by 2D[1H, 15N] NMR. When the platinum complex is in excess, the initial product, cis-[PtCl(NH3)2(Hmet-S)]+ undergoes slow ring closure to [Pt(NH3)2(Hmet-N,S)]2+. Slow ammine loss then occurs to give the isomer of [PtCl(NH3)(Hmet-N,S)]+ with chloride trans to sulfur. When methionine is in excess, a reaction sequence is proposed in which trans-[PtCl(NH3)(Hmet-S)2]+ isomerises to the cis-isomer, with subsequent ring closure reactions leading to cis-[Pt(Hmet-N,S)2]2+. Near pH 7, methionine is unreactive toward cis-[PtCl(OH)(NH3)2]. By contrast, L-cysteine reacts readily with cis-[PtCl(OH)(NH3)2] at pH 7, but there were many reaction products, including bridged species. Cis-[PtCl(OH)(NH3)2] reacts with reduced thiols in ultrafiltered plasma but these are oxidized if the plasma is not fresh or appropriately stored. With very low concentrations of the platinum complexes (35.5 microM), HPLC experiments (UV detection at 305 nm) indicate that the thiolate (probably cysteine) reactions become simpler as bridging becomes less important.

Crystal structures of a purple acid phosphatase, representing different steps of this enzyme's catalytic cycle

BackgroundPurple acid phosphatases belong to the family of binuclear metallohydrolases and are involved in a multitude of biological functions, ranging from bacterial killing and bone metabolism in animals to phosphate uptake in plants. Due to its role in bone resorption purple acid phosphatase has evolved into a promising target for the development of anti-osteoporotic chemotherapeutics. The design of specific and potent inhibitors for this enzyme is aided by detailed knowledge of its reaction mechanism. However, despite considerable effort in the last 10 years various aspects of the basic molecular mechanism of action are still not fully understood.ResultsRed kidney bean purple acid phosphatase is a heterovalent enzyme with an Fe(III)Zn(II) center in the active site. Two new structures with bound sulfate (2.4 Å) and fluoride (2.2 Å) provide insight into the pre-catalytic phase of its reaction cycle and phosphorolysis. The sulfate-bound structure illustrates the significance of an extensive hydrogen bonding network in the second coordination sphere in initial substrate binding and orientation prior to hydrolysis. Importantly, both metal ions are five-coordinate in this structure, with only one nucleophilic μ-hydroxide present in the metal-bridging position. The fluoride-bound structure provides visual support for an activation mechanism for this μ-hydroxide whereby substrate binding induces a shift of this bridging ligand towards the divalent metal ion, thus increasing its nucleophilicity.ConclusionIn combination with kinetic, crystallographic and spectroscopic data these structures of red kidney bean purple acid phosphatase facilitate the proposal of a comprehensive eight-step model for the catalytic mechanism of purple acid phosphatases in general.

Substrate-promoted formation of a catalytically competent binuclear center and regulation of reactivity in a glycerophosphodiesterase from Enterobacter aerogenes

The glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a promiscuous binuclear metallohydrolase that catalyzes the hydrolysis of mono-, di-, and triester substrates, including some organophosphate pesticides and products of the degradation of nerve agents. GpdQ has attracted recent attention as a promising enzymatic bioremediator. Here, we have investigated the catalytic mechanism of this versatile enzyme using a range of techniques. An improved crystal structure (1.9 A resolution) illustrates the presence of (i) an extended hydrogen bond network in the active site, and (ii) two possible nucleophiles, i.e., water/hydroxide ligands, coordinated to one or both metal ions. While it is at present not possible to unambiguously distinguish between these two possibilities, a reaction mechanism is proposed whereby the terminally bound H2O/OH(-) acts as the nucleophile, activated via hydrogen bonding by the bridging water molecule. Furthermore, the presence of substrate promotes the formation of a catalytically competent binuclear center by significantly enhancing the binding affinity of one of the metal ions in the active site. Asn80 appears to display coordination flexibility that may modulate enzyme activity. Kinetic data suggest that the rate-limiting step occurs after hydrolysis, i.e., the release of the phosphate moiety and the concomitant dissociation of one of the metal ions and/or associated conformational changes. Thus, it is proposed that GpdQ employs an intricate regulatory mechanism for catalysis, where coordination flexibility in one of the two metal binding sites is essential for optimal activity.

Lead macrocyclic complexes : the synthesis, complex formation and X-ray crystal structures of [Pb(L1)(NO3)2] and [Pb(L2)(NO3)2] (L1 = 1,4,7,10-tetraoxa-13-azacyclopentadecane, L2 = 1,4,7,10,13-pentaoxa-16-azacyclooctadecane)

Lead(II) complexes of the 15- and 18-membered ring macrocycles 1,4,7,10- tetraoxa-13-azacyclopentadecane (L1) and 1,4,7,10,13-pentaoxa-16-azacyclooctadecane (L2) have been prepared. The stability constants for the 1 : 1 lead complexes [L1, log β 6.0(1); L2, log β 8.4(1)] have been determined potentiometrically (0. 1 M NEt4ClO4, 95% methanol). The complexes [Pb(L1)(NO3)2] and [Pb(L2)(NO3)2] have been examined by 13C NMR spectroscopy and single-crystal X-ray structural analysis. In the molecule [Pb(L1)(NO3)2] the lead(II) cation is situated 1.52 A above the plane of the macrocyclic ring. The Pb-N(1) distance of 2.465(5) A is the shortest bond to lead(II) in the structure which also exhibits two short [2.627(4) and 2.643(4) A] and two long [2.909(4) and 2.992(5) A] PbOmacrocycle distances. The presence of a stereoactive lone pair of electrons on the cation is inferred from this stereochemistry. In [Pb(L2)(NO3)2] the lead(II) cation lies in the macrocyclic cavity. As for [Pb(L1)(NO3)2], the PbN(1) distance of 2.539(9) A is the shortest bond to lead in this structure, which has in addition two short [2.694(7) and 2.697(8) A] and three long [2.877(10), 2.951(6) and 2.999(9) A] PbOmacrocycle interactions. There is no evidence for a stereoactive lone pair of electrons in this structure.

The organophosphate-degrading enzyme from Agrobacterium radiobacter displays mechanistic flexibility for catalysis.

The OP (organophosphate)-degrading enzyme from Agrobacterium radiobacter (OpdA) is a binuclear metallohydrolase able to degrade highly toxic OP pesticides and nerve agents into less or non-toxic compounds. In the present study, the effect of metal ion substitutions and site-directed mutations on the catalytic properties of OpdA are investigated. The study shows the importance of both the metal ion composition and a hydrogen-bond network that connects the metal ion centre with the substrate-binding pocket using residues Arg254 and Tyr257 in the mechanism and substrate specificity of this enzyme. For the Co(II) derivative of OpdA two protonation equilibria (pKa1 ~5; pKa2 ~10) have been identified as relevant for catalysis, and a terminal hydroxide acts as the likely hydrolysis-initiating nucleophile. In contrast, the Zn(II) and Cd(II) derivatives only have one relevant protonation equilibrium (pKa ~4-5), and the μOH is the proposed nucleophile. The observed mechanistic flexibility may reconcile contrasting reaction models that have been published previously and may be beneficial for the rapid adaptation of OP-degrading enzymes to changing environmental pressures.

Metal-ion mutagenesis: Conversion of a purple acid phosphatase from sweet potato to a neutral phosphatase with the formation of an unprecedented catalytically competent MnIIMnII active site

The currently accepted paradigm is that the purple acid phosphatases (PAPs) require a heterovalent, dinuclear metal-ion center for catalysis. It is believed that this is an essential feature for these enzymes in order for them to operate under acidic conditions. A PAP from sweet potato is unusual in that it appears to have a specific requirement for manganese, forming a unique Fe(III)-mu-(O)-Mn(II) center under catalytically optimal conditions (Schenk et al. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 273). Herein, we demonstrate, with detailed electron paramagnetic resonance (EPR) spectroscopic and kinetic studies, that in this enzyme the chromophoric Fe(III) can be replaced by Mn(II), forming a catalytically active, unprecedented antiferromagnetically coupled homodivalent Mn(II)-mu-(H)OH-mu-carboxylato-Mn(II) center in a PAP. However, although the enzyme is still active, it no longer functions as an acid phosphatase, having optimal activity at neutral pH. Thus, PAPs may have evolved from distantly related divalent dinuclear metallohydrolases that operate under pH neutral conditions by stabilization of a trivalent-divalent metal-ion core. The present Mn(II)-Mn(II) system models these distant relatives, and the results herein make a significant contribution to our understanding of the role of the chromophoric metal ion as an activator of the nucleophile. In addition, the detailed analysis of strain broadened EPR spectra from exchange-coupled dinuclear Mn(II)-Mn(II) centers described herein provides the basis for the full interpretation of the EPR spectra from other dinuclear Mn metalloenzymes.

Synthesis and X-ray structural characterization of an iron(III) complex of the fluoroquinolone antimicrobial ciprofloxacin, [Fe(CIP)(NTA)]3·5H2O (NTANitrilotriacetato)

Abstract Reaction of the fluoroquinolone antimicrobial ciprofloxacin (cip) with iron(III) in the presence of nitrilotriacetate (nta) results in the isolation of yellow crystals of the complex [Fe(cip)(nta)]3·5H 2 O. The X-ray structural studies establish that, in the solid state, coordination of the iron(III) occurs through the keto and the carboxylic acid oxygen of the ciprofloxacin ligand to form a six-membered ring.

The divalent metal ion in the active site of uteroferrin modulates substrate binding and catalysis

The purple acid phosphatases (PAP) are binuclear metallohydrolases that catalyze the hydrolysis of a broad range of phosphomonoester substrates. The mode of substrate binding during catalysis and the identity of the nucleophile is subject to debate. Here, we used native Fe(3+)-Fe(2+) pig PAP (uteroferrin; Uf) and its Fe(3+)-Mn(2+) derivative to investigate the effect of metal ion substitution on the mechanism of catalysis. Replacement of the Fe(2+) by Mn(2+) lowers the reactivity of Uf. However, using stopped-flow measurements it could be shown that this replacement facilitates approximately a ten-fold faster reaction between both substrate and inorganic phosphate with the chromophoric Fe(3+) site. These data also indicate that in both metal forms of Uf, phenyl phosphate hydrolysis occurs faster than formation of a mu-1,3 phosphate complex. The slower rate of interaction between substrate and the Fe(3+) site relative to catalysis suggests that the substrate is hydrolyzed while coordinated only to the divalent metal ion. The likely nucleophile is a water molecule in the second coordination sphere, activated by a hydroxide terminally coordinated to Fe(3+). The faster rates of interaction with the Fe(3+) site in the Fe(3+)-Mn(2+) derivative than the native Fe(3+)-Fe(2+) form are likely mediated via a hydrogen bond network connecting the first and second coordination spheres, and illustrate how the selection of metal ions may be important in fine-tuning the function of this enzyme.

Monoesterase Activity of a Purple Acid Phosphatase Mimic with a Cyclam Platform

The synthesis and characterization of a novel dinucleating ligand L (L=4,11-dimethyl-1,8-bis{2-[N-(di-2-pyridylmethyl)amino]ethyl}cyclam) and its μ-oxo-bridged diferric complex [(H(2)L){Fe(III)(2)(O)}(Cl)(4)](2+) are reported. This diiron(III) complex is the first example of a truly functional purple acid phosphatase (PAP) mimic as it accelerates the hydrolysis of the activated phosphomonoester 2,4-dinitrophenyl phosphate (DNPP). The spectroscopic and kinetic data indicate that only substrates that are monodentately bound to one of the two ferric ions can be attacked by a suitable nucleophile. This is, most probably, a terminal iron(III)-bound hydroxide. DFT calculations support this assumption and also highlight the importance of secondary interactions, exerted by the protonated cyclam platform, for the positioning and activation of the iron(III)-bound substrate. Similar effects are postulated in the native enzyme but addressed in PAP mimics for the first time.

Structural flexibility enhances the reactivity of the bioremediator glycerophosphodiesterase by fine-tuning its mechanism of hydrolysis.

The glycerophosphodiesterase from Enterobacter aerogenes (GpdQ) belongs to the family of binuclear metallohydrolases and has attracted recent attention due to its potential in bioremediation. Formation of a catalytically competent binuclear center is promoted by the substrate (Hadler et al. J. Am. Chem. Soc. 2008, 130, 14129). Using the paramagnetic properties of Mn(II), we estimated the K(d) values for the metal ions in the alpha and beta sites to be 29 and 344 microM, respectively, in the absence of a substrate analogue. In its presence, the affinity of the beta site increases substantially (K(d) = 56 microM), while that of the alpha site is not greatly affected (K(d) = 17 microM). Stopped-flow fluorescence measurements identified three distinct phases in the catalytic turnover, associated with the initial binding of substrate to the active site (k(obs1)), the assembly of a catalytically active binuclear center (k(obs2)), and subsequent slower structural rearrangements to optimize catalysis (k(obs3)). These three phases depend on the concentration of substrate ([S]), with k(obs1) and k(obs2) reaching maximum values at high [S] (354 and 38 s(-1), respectively), whereas k(obs3) is reduced as [S] is increased. The k(cat) for the hydrolysis of the substrate bis(para-nitrophenyl) phosphate (approximately 1 s(-1)) gradually increases from the moment of initiating the reaction, reaching a maximum when the structural change associated with k(obs3) is complete. This structural change is mediated via an extensive hydrogen-bond network that connects the coordination sphere with the substrate binding pocket, as demonstrated by mutation of two residues in this network (His81 and His217). The identities of both the substrate and the metal ion also affect interactions within this H-bond network, thus leading to some mechanistic variations. Overall, the mechanism employed by GpdQ is a paradigm of a substrate- and metal-ion-induced fit to optimize catalysis.