Binding of sulfonamide and acetamide to the active-site Zn2+ in carbonic anhydrase: a theoretical study

Self-consistent field molecular orbital (SCF MO) calculations at both 4-31G and STO-3G levels have been used to examine the binding conformations of sulfonamide and acetamide compounds to the active site of carbonic anhydrase. The results are as follows: (1) sulfonamide binds to the Zn2+ ion in its deprotonated form through the sulfonamide nitrogen to the fourth coordination site of the metal ion; (2) acetamide as neutral species binds to the basic form of the enzyme through the carbonyl oxygen to the fifth coordination site of the metal ion; and (3) the acetamidate ion binds to the acid form of the enzyme through the amide nitrogen to form a tetracoordinated metal complex with three histidine ligands. Analysis of the effects of individual active-site residues on the binding conformations of these inhibitors suggests that metal alone favors bidentate coordination of sulfonamidate and acetamidate complexes and that electron donation from three histidine ligands to the metal ion determines the formation of a tetracoordinated metal complex, which is further stabilized by the presence of Thr 199, as it receives one hydrogen bond from the sulfonamide NH- or from the acetamide NH- and donates a backbone NH hydrogen bond to a sulfonamide oxygen. The calculated binding conformation of sulfonamide and the hydrogen-bonding interactions between sulfonamide and the enzyme are consistent with the X-ray diffraction study of the AMSulf-HCA II complex. However, no X-ray structures are available for amide-HCA II complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of the metal ion in the refolding of denatured bovine Co(II)-carbonic anhydrase II

Conditions for reactivation of guanidine-HCl-denatured bovine Co(II)-carbonic anhydrase II are given. The renaturation is accompanied by recovery of the native Co(II)-spectrum of the enzyme. After studying the kinetics of the renaturation process, the metal ion involvement in the refolding pathway can be summarized as follows: (1) Formation of an inactive Co(II)-intermediate with the metal ion firmly bound. No native Co(II)-spectrum is observed in this state, probably due to octahedral coordination of the metal ion in this intermediate. (2) Formation of an inactive Co(II)-intermediate with a native Co(II)-spectrum. The final tetrahedral coordination of the metal ion seems to have been formed in this state. (3) Formation of the active conformation of the enzyme. A functioning active-site is formed after some rearrangements of the polypeptide chain. This isomerisation step does not need to be preceded by formation of the intermediate with a native Co(II)-spectrum. Coordination of Co2+ in a native-like manner is, however, a prerequisite for enzymic activity. It is tentatively suggested that the metal ion is involved in stabilizing a nucleation structure formed at the bottom of the active centre. This probably occurs through binding of Co2+ to some or all of its histidyl ligands in this region after an early structuration of the metal ion binding site. The mechanisms of Co2+ appear to be similar for the refolding enzyme and the native apoenzyme, inferring that the binding site formed as a result of the nucleation process probably has the same structure as in the native conformation.     

Dissociation of outer-sphere water is rate-limiting for the binding of ligands in the active site of horse liver alcohol dehydrogenase

The association of imidazole and auramine O to native horse-liver alcohol dehydrogenase [Zn(II)LADH] and active-site specifically cobalt(II)-substituted horse-liver alcohol dehydrogenase [Co(II)LADH], respectively, has been investigated. In all cases [except imidazole binding to Zn(II)LADH in the presence of auramine O] the association rates approached an upper limit (kmax). The kmax values were compared for the metal ligands imidazole (monodentate), 1,10-phenanthroline and 2,2'-bipyridine (bidentate; see also the preceding paper), and for auramine O which does not coordinate to the catalytic metal ion. Independent of the large differences in their structure and metal-bonding capability, all these compounds exhibit common, maximum, limiting rate constants of about 60 s-1 and 200 s-1 for Co(II)LADH and Zn(II)LADH, respectively. These results demonstrate that kmax is strongly dependent on the catalytic metal ion but not on the ligand. The absence of spectral changes in the d-d transitions of the catalytic Co(II) ion upon auramine O binding to Co(II)LADH indicates that the rate-limiting step is not accompanied by a major conformational change. Finally, it is concluded that reactions in the inner coordination sphere of the catalytic metal ion (i.e. the metal-bound water molecule) are not responsible for the step characterized by kmax. We propose the rate-limiting step to consist of the dissociation of one or several water molecules from the second coordination sphere of the catalytic metal ion in the active site of LADH in its open conformation.     

Electronic spectroscopy of cobalt angiotensin converting enzyme and its inhibitor complexes

Zinc, the catalytically essential metal of angiotensin converting enzyme (ACE), has been replaced by cobalt(II) to give an active, chromophoric enzyme that is spectroscopically responsive to inhibitor binding. Visible absorption spectroscopy and magnetic circular dichroic spectropolarimetry have been used to characterize the catalytic metal binding site in both the cobalt enzyme and in several enzyme-inhibitor complexes. The visible absorption spectrum of cobalt ACE exhibits a single broad maximum (525 nm) of relatively low absorptivity (epsilon = 75 M-1 cm-1). In contrast, the spectra of enzyme-inhibitor complexes display more clearly defined maxima at longer wavelengths (525-637 nm) and of markedly higher absorptivities (130-560 M-1 cm-1). The large spectral response indicates that changes in the cobalt ion coordination sphere occur on inhibitor binding. Magnetic circular dichroic spectropolarimetry has shown that the metal coordination geometry in the inhibitor complexes is tetrahedral and of higher symmetry than in cobalt ACE alone. The presence of sulfur----cobalt charge-transfer bands in both the visible absorption and magnetic circular dichroic spectra of the cobalt ACE-Captopril complex confirm direct ligation of the thiol group of the inhibitor to the active-site metal.     

Interaction of sulphate and chloride with cobalt(II)-carbonic anhydrase

The interaction between Cobalt(II)-Bovine Carbonic Anhydrase II and the inhibitors sulphate and chloride have been investigated through 1H NMR and electronic absorption spectroscopies. Both inhibitors bind to the metal ion forming a 1:1 adduct and the corresponding affinity constants have been determined. These inhibitors interact weakly with CoBCA II and this interaction only occurs at low pH values. The T1 values of the meta-like protons of the coordinated histidines have been measured. The coordination number of the metal ion in the adducts is discussed on the basis of temperature dependence of the isotropic shifts, T1, and molar absorbance values.     

Preparation by direct metal exchange and kinetic study of active site metal substituted class I and class II Clostridium histolyticum collagenases

Active site metal substitutions for both gamma- and zeta-collagenases from Clostridium histolyticum have been made by direct metal exchange. The incubation of Co(II), Cu(II), Ni(II), Cd(II), and Hg(II) with these native collagenases results in changes in activity that parallel those observed for the reconstitution of the respective apoenzymes with these metal ions. For both collagenases, the exchange reactions with Co(II) and Cu(II) are complete within 1 min. However, the changes in activity observed on addition of Ni(II), Cd(II), and Hg(II) to gamma-collagenase and Cd(II) and Hg(II) to zeta-collagenase are time dependent. The kinetic parameters Kcat and KM have been determined for each of the active metallospecies. The substitution of the active-site metal ion in gamma-collagenase results in changes in both kcat and KM, while the effect observed in zeta-collagenase is primarily on KM. This suggests that there are differences in the mechanisms of these two collagenases, at least with respect to the role of the zinc ion in catalysis.     

Crystallographic studies of the binding of protonated and unprotonated inhibitors to carbonic anhydrase using hydrogen sulphide and nitrate anions.

The structures of human carbonic-anhydrase-II complexes with the anionic inhibitors hydrogen sulphide (HS-) and nitrate (NO3-) have been determined by X-ray diffraction at 0.19-nm resolution from crystals soaked at pH 7.8 and 6.0, respectively. The modes of binding of these two anions differ markedly from each other. The strong inhibitor HS- replaces the native zinc-bound water/hydroxide (Wat263) leaving the tetrahedral metal geometry unaltered and acts as a hydrogen-bonding donor towards Thr199 gamma. The weak NO3- inhibitor does not displace Wat263 from the metal coordination but occupies a fifth binding site changing the zinc coordination polyhedron into a slightly distorted trigonal bipyramid. The interaction of NO3- with the metal is weak; the nearest of its oxygen atoms being at a distance of 0.28 nm from the zinc ion. The binding of nitrate to the enzyme is completed by a hydrogen bond to the metal coordinated Wat263 and a second one to a water molecule of the active-site cavity. The structures of the two complexes help to rationalize the binding of anionic inhibitors to carbonic anhydrase and the binding mode displayed by NO39 may be relevant to the catalytic mechanism.     

pK values for active site residues of carboxypeptidase A

The phenolic group of active site residue Tyr-248 in carboxypeptidase A has a pKa value of 10.06, as determined from the pH dependence of its rate of nitration by tetranitromethane. The decrease in enzyme activity (kcat/Km) in alkaline solution, characterized by a pKa value of approximately 9.0 (for cobalt carboxypeptidase A), is associated with the protonation state of an imidazole ligand of the active-site metal ion, as indicated by a selective pH dependence of the 1H NMR spectrum of the enzyme. Inhibition of the cobalt-substituted enzyme by 2-(1-carboxy-2-phenylethyl)phenol and its 4,6-dichloro- and 4-phenylazo-derivatives confirms that the decrease in enzyme activity (kcat/Km) in acidic solution, characterized by a pKa value of 5.8, is due to the protonation state of a water molecule bound to the active-site metal ion in the absence of substrate. Changes in the coordination number of the active-site metal ion are seen in its visible absorption spectrum as a consequence of binding of the phenolic inhibitors. Conventional concepts regarding the mechanisms of the enzyme are brought into question.     

Inhibition of glutamate carboxypeptidase II by phosphonamidothionate derivatives of glutamic acid

A limited series of N-thiophosphonyl-glutamates were found to be inhibitors of the prostate-specific membrane antigen (PSMA) form of glutamate carboxypeptidase II. Comparative inhibitory profiles of an analogous O-thiophosphonyl-2-hydroxyglutarate revealed that the amido-linkage of the N-thiophosphonyl-glutamate provides a significant enhancement of inhibitory potency presumably due to significant hydrogen-bonding interactions with acceptor groups in the active-site of PSMA resulting in tighter binding. An analogous N-phosphonyl-glutamate exhibited significantly greater inhibitory potency than the parent N-thiophosphonyl-glutamate indicating that the sulfur ligand of the N-thiophosphonyl-glutamates is responsible for less favorable active-site interactions than oxygen, potentially due to steric crowding from the longer P-S bond or as a result of active-site metal substitution of Co(II) for Zn(II) arising from assay conditions.     

The reaction mechanism of the Ga(III)Zn(II) derivative of uteroferrin and corresponding biomimetics

Purple acid phosphatase from pig uterine fluid (uteroferrin), a representative of the diverse family of binuclear metallohydrolases, requires a heterovalent Fe(III)Fe(II) center for catalytic activity. The active-site structure and reaction mechanism of this enzyme were probed with a combination of methods including metal ion replacement and biomimetic studies. Specifically, the asymmetric ligand 2-bis{[(2-pyridylmethyl)-aminomethyl]-6-[(2-hydroxybenzyl)(2-pyridylmethyl)]aminomethyl}-4-methylphenol and two symmetric analogues that contain the softer and harder sites of the asymmetric unit were employed to assess the site selectivity of the trivalent and divalent metal ions using (71)Ga NMR, mass spectrometry and X-ray crystallography. An exclusive preference of the harder site of the asymmetric ligand for the trivalent metal ion was observed. Comparison of the reactivities of the biomimetics with Ga(III)Zn(II) and Fe(III)Zn(II) centers indicates a higher turnover for the former, suggesting that the M(III)-bound hydroxide acts as the reaction-initiating nucleophile. Catalytically active Ga(III)Zn(II) and Fe(III)Zn(II) derivatives were also generated in the active site of uteroferrin. As in the case of the biomimetics, the Ga(III) derivative has increased reactivity, and a comparison of the pH dependence of the catalytic parameters of native uteroferrin and its metal ion derivatives supports a flexible mechanistic strategy whereby both the mu-(hydr)oxide and the terminal M(III)-bound hydroxide can act as nucleophiles, depending on the metal ion composition, the geometry of the second coordination sphere and the substrate.     

A thiono-beta-lactam substrate for the beta-lactamase II of Bacillus cereus. Evidence for direct interaction between the essential metal ion and substrate.

An 8-thionocephalosporin was shown to be a substrate of the beta-lactamase II of Bacillus cereus, a zinc metalloenzyme. Although it is a poorer substrate, as judged by the Kcat./Km parameter, than the corresponding 8-oxocephalosporin, the discrimination against sulphur decreased when the bivalent metal ion in the enzyme active site was varied in the order Mn2+ (the manganese enzyme catalysed the hydrolysis of the oxo compound but not that of the thiono compound), Zn2+, Co2+ and Cd2+. This result is taken as evidence for kinetically significant direct contact between the active-site metal ion of beta-lactamase II and the beta-lactam carbonyl heteroatom. No evidence was obtained, however, for accumulation of an intermediate with such co-ordination present.     

Uncoupling metallonuclease metal ion binding sites via nudge mutagenesis

The hydrolysis of phosphodiester bonds by nucleases is critical to nucleic acid processing. Many nucleases utilize metal ion cofactors, and for a number of these enzymes two active-site metal ions have been detected. Testing proposed mechanistic roles for individual bound metal ions has been hampered by the similarity between the sites and cooperative behavior. In the homodimeric PvuII restriction endonuclease, the metal ion dependence of DNA binding is sigmoidal and consistent with two classes of coupled metal ion binding sites. We reasoned that a conservative active-site mutation would perturb the ligand field sufficiently to observe the titration of individual metal ion binding sites without significantly disturbing enzyme function. Indeed, mutation of a Tyr residue 5.5 A from both metal ions in the enzyme-substrate crystal structure (Y94F) renders the metal ion dependence of DNA binding biphasic: two classes of metal ion binding sites become distinct in the presence of DNA. The perturbation in metal ion coordination is supported by 1H-15N heteronuclear single quantum coherence spectra of enzyme-Ca(II) and enzyme-Ca(II)-DNA complexes. Metal ion binding by free Y94F is basically unperturbed: through multiple experiments with different metal ions, the data are consistent with two alkaline earth metal ion binding sites per subunit of low millimolar affinity, behavior which is very similar to that of the wild type. The results presented here indicate a role for the hydroxyl group of Tyr94 in the coupling of metal ion binding sites in the presence of DNA. Its removal causes the affinities for the two metal ion binding sites to be resolved in the presence of substrate. Such tuning of metal ion affinities will be invaluable to efforts to ascertain the contributions of individual bound metal ions to metallonuclease function.     

Function of His185 in Aquifex aeolicus 3-deoxy-D-manno-octulosonate 8-phosphate synthase

Aquifex aeolicus 3-deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the condensation of arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) by favoring the activation of a water molecule coordinated to the active-site metal ion. Cys11, His185, Glu222 and Asp233 are the other metal ligands. Wild-type KDO8PS is purified with Zn(2+) or Fe(2+) in the active site, but maximal activity in vitro is achieved when the endogenous metal is replaced with Cd(2+). The H185G enzyme retains 8% of the wild-type activity. ICP mass spectrometry analysis indicates that loss of His185 decreases the enzyme affinity for Fe(2+), but not for Zn(2+). However, maximal activity is again achieved by substitution of the endogenous metal with Cd(2+). We have determined the X-ray structures of the Cd(2+) H185G enzyme in its substrate-free form, and in complex with PEP, and PEP plus A5P. These structures show a normal amount of Cd(2+) bound, suggesting that coordination by His185 is not essential to retain Cd(2+) in the active site. Nonetheless, there are significant changes in the coordination sphere of Cd(2+) with respect to the wild-type enzyme, as the carboxylate moiety of PEP binds directly to the metal ion and replaces water and His185 as ligands. These observations indicate that the primary function of His185 in A.aeolicus KDO8PS is to orient PEP in the active site of the enzyme in such a way that a water molecule on the sinister (si) side of PEP can be activated by direct coordination to the metal ion.     

1H NMR spectroscopic characterization of binary and ternary complexes of cobalt(II) carboxypeptidase A with inhibitors

The binding of L- and D-phenylalanine and carboxylate inhibitors to cobalt(II)-substituted carboxypeptidase A, Co(II)CPD (E), in the presence and absence of pseudohalogens (X = N3-, NCO-, and NCS-) has been studied by 1H NMR spectroscopy. This technique monitors the proton signals of histidine residues bound to cobalt(II) and is therefore sensitive to the interactions of inhibitors that perturb the coordination sphere of the metal. Enzyme-inhibitor complexes, E.I, E.I2, and E.I.X, each with characteristic NMR features, have been identified. Thus, for example, L-Phe binds close to the metal ion to form a 1:1 complex, whereas D-Phe binds stepwise, first to a nonmetal site and then to the metal ion to form a 2:1 complex. Both acetate and phenylacetate also form 2:1 adducts stepwise with the enzyme, but beta-phenylpropionate gives a 2:1 complex without any detectable 1:1 intermediate. N3-, NCO-, and NCS- generate E.I.X ternary complexes directly with Co(II)CPD.L-Phe and indirectly with the D-Phe and carboxylate inhibitor 2:1 complexes by displacing the second moiety from its metal binding site. The NMR data suggest that when the carboxylate group of a substrate or inhibitor binds at the active site, a conformational change occurs that allows a second ligand molecule to bind to the metal ion, altering its coordination sphere and thereby attenuating the bidentate behavior of Glu-72. The 1H NMR signals also reflect alterations in the histidine interactions with the metal upon inhibitor binding. Isotropic shifts in the signals for the C-4 (c) and N protons (a) of one of the histidine ligands are readily observed in all of these complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystallographic studies of metal ion-DNA interactions: different binding modes of cobalt(II), copper(II) and barium(II) to N7 of guanines in Z-DNA and a drug-DNA complex.

Metal ion coordination to nucleic acids is not only required for charge neutralization, it is also essential for the biological function of nucleic acids. The structural impact of different metal ion coordinations of DNA helices is an open question. We carried out X-ray diffraction analyses of the interactions of the two transition metal ions Co(II) and Cu(II) and an alkaline earth metal ion Ba(II), with DNA of different conformations. In crystals, Co(II) ion binds exclusively at the N7 position of guanine bases by direct coordination. The coordination geometry around Co(II) is octahedral, although some sites have an incomplete hydration shell. The averaged Co-N7 bond distance is 2.3 A. The averaged Co-N7-C8 angle is 121 degrees, significantly smaller than the value of 128 degrees if the Co-N7 vector were to bisect the C5-N7-C8 bond angle. Model building of Co(II) binding to guanine N7 in B-DNA indicates that the coordinated waters in the axial positions would have a van der Waals clash with the neighboring base on the 5' side. In contrast, the major groove of A-DNA does not have enough room to accommodate the entire hydration shell. This suggests that Co(II) binding to either B-DNA or A-DNA may induce significant conformational changes. The Z-DNA structure of Cu(II)-soaked CGCGTG crystal revealed that the Cu(II) ion is bis-coordinated to N7 position of G10 and #G12 (# denotes a symmetry-related position) bases with a trigonal bipyramid geometry, suggesting a possible N7-Cu-N7 crosslinking mechanism. A similar bis-coordination to two guanines has also been seen in the interaction of Cu(II) in m5CGUAm5CG Z-DNA crystal and of Ba(II) with two other Z-DNA crystals.     

Inhibition of carboxypeptidase A by D-penicillamine: mechanism and implications for drug design

Zinc metalloprotease inhibitors are usually designed to inactivate the enzyme by forming a stable ternary complex with the enzyme and active-site zinc. D-Cysteine inhibits carboxypeptidase, ZnCPD, by forming such a complex, with a K(i) of 2.3 microM. In contrast, the antiarthritis drug D-penicillamine, D-PEN, which differs from D-Cys only by the presence of two methyl groups on the beta-carbon, inhibits ZnCPD by promoting the release of the active-site zinc. We have given the name catalytic chelator to such inhibitors. Inhibition is a two-step process characterized by formation of a complex with the enzyme (K(i(initial)) = 1.2 mM) followed by release of the active-site zinc at rates up to 420-fold faster than the spontaneous release. The initial rate of substrate hydrolysis at completion of the second step also depends on D-PEN concentration, reflecting formation of a thermodynamic equilibrium governed by the stability constants of chelator and apocarboxypeptidase for zinc (K(i(final)) = 0.25 mM). The interaction of D-PEN and D-Cys with the active-site metal has been examined by replacing the active-site zinc by a chromophoric cobalt atom. Both inhibitors perturb the d-d transitions of CoCPD in the 500-600 nm region within milliseconds of mixing but only the CoCPD.D-Cys complex displays a strong S --> Co(II) charge-transfer band at 340 nm indicative of a metal-sulfur bond. While the D-Cys complex is stable, the CoCPD.D-PEN complex breaks down to apoenzyme and Co(D-PEN)(2) with a half-life of 0.5 s. D-PEN is the first drug found to inhibit a metalloprotease by increasing the dissociation rate constant of the active-site metal. The ability of D-PEN to catalyze metal removal from carboxypeptidase A and other zinc proteases suggests a possible mechanism of action in arthritis and Wilson's disease and may also underlie complications associated with its clinical use.     

Phospholipid-deacylating enzymes of rabbit platelets.

The inhibition of selenium-glutathione peroxidase by metal ions was studied by means of a direct spectrophotometric assay that monitors at 237 nm the decrease of GS^? concentration with time. Cadmium (II) and zinc (II) ions were the most potent inhibitors, while silver (I), mercury (II), cobalt (II), and lead (II) inhibited to a lesser extent. Inhibition by these metal ions was competitive with respect to the donor substrate, GSH. Competitive inhibition was verified for cadmium (II) ion by means of an assay employing Ellman's reagent, 5,5′-dithiobis-2-nitrobenzoic acid. Inhibition by cadmium (II) ion was noncompetitive with respect to the acceptor substrate, t-butyl hydroperoxide. Inhibitor constants obtained from Lineweaver-Burk plots and binding constants obtained from Scatchard plots were comparable. Correlation of inhibitor constants with chemical and physical properties showed a dependence on the softness of the metal ion as an acid and also a dependence on ionic size.     

Analyzing the binding of Co(II)-specific inhibitors to the methionyl aminopeptidases from Escherichia coli and Pyrococcus furiosus

Methionine aminopeptidases (MetAPs) represent a unique class of protease that is capable of the hydrolytic removal of an N-terminal methionine residue from nascent polypeptide chains. MetAPs are physiologically important enzymes; hence, there is considerable interest in developing inhibitors that can be used as antiangiogenic and antimicrobial agents. A detailed kinetic and spectroscopic study has been performed to probe the binding of a triazole-based inhibitor and a bestatin-based inhibitor to both Mn(II)- and Co(II)-loaded type-I (Escherichia coli) and type-II (Pyrococcus furiosus) MetAPs. Both inhibitors were found to be moderate competitive inhibitors. The triazole-type inhibitor was found to interact with both active-site metal ions, while the bestatin-type inhibitor was capable of switching its mode of binding depending on the metal in the active site and the type of MetAP enzyme.     

Investigation of the catalytic mechanism of yeast inorganic pyrophosphatase

P1,P2-Bidentate Co(NH3)4PP was found to be a competitive inhibitor of pyrophosphatase vs. MgPP (Kis = 8.7 mM, pH 7) and, in the presence of Mg2+, an active substrate as well. P1,P2-Bidentate Cr(III) complexes of pyrophosphate, imidodiphosphate, and methylenediphosphonate were also competitive inhibitors vs. MgPP (pH 5.9; Kis = 0.2, 0.2, and 0.4 mM, respectively). In the presence of Mg2+, P1,P2-bidentate Cr(H2O)4PP was found to have a Km 10-fold greater and a turnover number 36-fold smaller than MgPP at pH 5.9. Mg2+, Mn2+, Co2+, Zn2+, Cd2+, Ni2+, and Fe2+ activate the CrPP--pyrophosphatase reaction, while Ca2+ and Ba2+ are not activators but serve as competitive inhibitors vs. Mg2+ (Kis = 0.35 and 2.3 mM). At levels above 0.1 mM, Mn2+, Co2+, and Zn2+ show activator inhibition. Kinetic studies with CrPP and Mg2+ suggest that the kinetic mechanism is rapid equilibrium ordered, with CrPP adding before Mg2+. pH studies of the MgPP/Mg2+ reaction and the CrPP/Mg2+ reaction suggest that the active form of the substrate is (MgPP)2- and that the uncomplexed metal ion cofactor interacts with at least two active-site residues, one possibly via H bonding and the other by direct coordination. The former group (pKa = 5.6) appears on the basis of temperature and solvent perturbation studies to be a carboxylic acid. The MgPP reaction also requires that an active-site residue (pKa = 7.5) be protonated. Temperature and solvent perturbation studies suggest that this residue is an amine. A mechanism accounting for these observations is presented.