Rate-limiting Proton Transfer Research Articles

A non-canonical nucleophile unlocks a new mechanistic pathway in a designed enzyme

Directed evolution of computationally designed enzymes has provided new insights into the emergence of sophisticated catalytic sites in proteins. In this regard, we have recently shown that a histidine nucleophile and a flexible arginine can work in synergy to accelerate the Morita-Baylis-Hillman (MBH) reaction with unrivalled efficiency. Here, we show that replacing the catalytic histidine with a non-canonical Nδ-methylhistidine (MeHis23) nucleophile leads to a substantially altered evolutionary outcome in which the catalytic Arg124 has been abandoned. Instead, Glu26 has emerged, which mediates a rate-limiting proton transfer step to deliver an enzyme (BHMeHis1.8) that is more than an order of magnitude more active than our earlier MBHase. Interestingly, although MeHis23 to His substitution in BHMeHis1.8 reduces activity by 4-fold, the resulting His containing variant is still a potent MBH biocatalyst. However, analysis of the BHMeHis1.8 evolutionary trajectory reveals that the MeHis nucleophile was crucial in the early stages of engineering to unlock the new mechanistic pathway. This study demonstrates how even subtle perturbations to key catalytic elements of designed enzymes can lead to vastly different evolutionary outcomes, resulting in new mechanistic solutions to complex chemical transformations.

Facilitating Molecular Activation and Proton Feeding by Dual Active Sites on Polymeric Carbon Nitride for Efficient CO2 Photoreduction.

Photoreduction of CO2 provides an appealing way to alleviate the energy crisis and manage the global carbon balance but is limited by the high activation energy and the rate-limiting proton transfer. We now develop a dual-site strategy for high-efficiency CO2 conversion through polarizing CO2 molecules at pyridine N vacancies and accelerating the intermediate protonation by protonated pyridine N adjacent to nitrogen vacancies on polymeric carbon nitride. Our photocatalysts with atomic-level engineered active sites manifest a high CO production rate of 1835 μmol g-1 h-1 , 183 times higher than the pristine bulk carbon nitride. Theoretical prediction and experimental studies confirm that such excellent performance is attributed to the synergistic effect between vacant and protonated pyridine N in decreasing the formation energy of the key *COOH intermediates and the efficient electron transfer relay facilitated by the defect-induced shallow trap state and homogeneous charge mediators.

Role of High-Spin Species and Pendant Amines in Electrocatalytic Alcohol Oxidation by a Nickel Phosphine Complex

The development of active and efficient electrocatalysts for oxidation of alcohols using earth-abundant metals will aid progress toward a renewable energy economy. Here, we present a detailed mechanistic study of electrocatalytic benzyl alcohol oxidation by a molecular nickel complex containing pendant amines using a combination of kinetic studies, nuclear magnetic resonance spectroscopy, and density functional theory. The catalyst preferentially binds alcohol in a high-spin octahedral geometry, but this complex is not readily deprotonated by an exogenous base and inhibits catalysis. Dissociation of one or more solvent ligands returns the complex to a low-spin state that can be deprotonated. Kinetic modeling indicates that the off-cycle high-spin intermediate lowers the catalytic turnover frequency by a factor of 8, suggesting that substantial gains in activity can be attained by improvements to the catalyst coordination geometry. In a second finding, we demonstrate that the pendant amine of the catalyst only functions as a proton relay for the potential-determining step, oxidation of a nickel hydride intermediate, but does not have a substantial impact on the overall rate for oxidation of benzyl alcohol to benzaldehyde. This result contradicts the common expectation for pendant amines to participate in rate-limiting proton transfer reactions.

Evidence of Rate Limiting Proton Transfer in an SNAr Aminolysis in Acetonitrile under Synthetically Relevant Conditions.

An early synthetic step in the synthesis of adavosertib, AZD1775, is the SNAr reaction between 4-fluoronitrobenzene and 1-methylpiperazine in acetonitrile. A simple kinetics-based design of four reaction profiling experiments was used to investigate the kinetics of the reaction for the purpose of building a kinetic model. Fitting of the reaction profile data from two experiments conducted at 70 °C with a different excess of 1-methylpiperazine showed the reaction to follow a third-order rate law with a second-order dependence upon 1-methylpiperazine. This was rationalized in terms of the reaction following a rate-limiting proton transfer mechanism (base catalyzed) in which the progress to product is driven by a proton transfer involving a second molecule of 1-methylpiperazine. The experimentally determined entropy of activation of -180 J K-1 is consistent with this mechanism. The formation of a low level impurity was found to be due to the presence of traces of piperazine in the 1-methylpiperazine, which was shown to react approximately 15 times faster than 1-methylpiperazine at 70 °C. The rate constants for the 1-methylpiperazine catalyzed reaction of piperazine, 1-methylpiperazine, and the piperazine derived impurity were found to correlate in a Brønsted type analysis with the pKa's (acetonitrile) of the amine nucleophile.

Semiempirical method for examining asynchronicity in metal–oxido-mediated C–H bond activation

The oxidation of substrates via the cleavage of thermodynamically strong C-H bonds is an essential part of mammalian metabolism. These reactions are predominantly carried out by enzymes that produce high-valent metal-oxido species, which are directly responsible for cleaving the C-H bonds. While much is known about the identity of these transient intermediates, the mechanistic factors that enable metal-oxido species to accomplish such difficult reactions are still incomplete. For synthetic metal-oxido species, C-H bond cleavage is often mechanistically described as synchronous, proton-coupled electron transfer (PCET). However, data have emerged that suggest that the basicity of the M-oxido unit is the key determinant in achieving enzymatic function, thus requiring alternative mechanisms whereby proton transfer (PT) has a more dominant role than electron transfer (ET). To bridge this knowledge gap, the reactivity of a monomeric MnIV-oxido complex with a series of external substrates was studied, resulting in a spread of over 104 in their second-order rate constants that tracked with the acidity of the C-H bonds. Mechanisms that included either synchronous PCET or rate-limiting PT, followed by ET, did not explain our results, which led to a proposed PCET mechanism with asynchronous transition states that are dominated by PT. To support this premise, we report a semiempirical free energy analysis that can predict the relative contributions of PT and ET for a given set of substrates. These findings underscore why the basicity of M-oxido units needs to be considered in C-H functionalization.

Reversible nickel-metallacycle formation with a phosphinimine-based pincer ligand.

Pincer ligands have a remarkable ability to impart control over small molecule activation chemistry and catalytic activity; therefore, the design of new pincer ligands and the exploration of their reactivity profiles continues to be a frontier in synthetic inorganic chemistry. In this work, a novel, monoanionic NNN pincer ligand containing two phosphinimine donors was used to create a series of mononuclear Ni complexes. Ligand metallation in the presence of NaOPh yielded a nickel phenoxide complex that was used to form a mononuclear hydride complex on treatment with pinacolborane. Attempts at ligand metallation with NaN(SiMe3)2 resulted in the activation of both phosphinimine methyl groups to yield an anionic, cis-dialkyl product, in which dissociation of one phosphinimine nitrogen leads to retention of a square planar coordination environment about Ni. Protonolysis of this dialkyl species generated a monoalkyl product that retained the 4-membered metallacycle. The insertion of 2,6-dimethylphenyl isocyanide (xylNC) into this nickel metallacycle, followed by proton transfer, generated a new five-membered nickel metallacycle. Kinetic studies suggested rate-limiting proton transfer (KIE ≥ 3.9 ± 0.5) from the α-methylene unit of the putative iminoacyl intermediate.

Leaving Group Effects in a Series of Electrosprayed CcHhN1 Anthracene Derivatives.

We investigate the gas-phase structures and fragmentation pathways of model compounds of anthracene derivatives of the general formula CcHhN1 utilizing tandem mass spectrometry and computational methods. We vary the substituent alkyl chain length, composition, and degree of branching. We find substantial experimental and theoretical differences between the linear and branched congeners in terms of fragmentation thresholds, available pathways, and distribution of products. Our calculations predict that the linear substituents initially isomerize to form lower energy branched isomers prior to loss of the alkyl substituents as alkenes. The rate-determining chemistry underlying these related processes is dominated by the ability to stabilize the alkene loss transition structures. This task is more effectively undertaken by branched substituents. Consequently, analyte lability systematically increased with degree of branching (linear < secondary < tertiary). The resulting anthracen-9-ylmethaniminium ion generated from these alkene loss reactions undergoes rate-limiting proton transfer to enable expulsion of either hydrogen cyanide or CNH. The combination of the differences in primary fragmentation thresholds and degree of radical-based fragmentation processes provide a potential means of distinguishing compounds that contain branched alkyl chain substituents from those with linear ones.

Regulating the Basicity of Metal-Oxido Complexes with a Single Hydrogen Bond and Its Effect on C-H Bond Cleavage.

The functionalization of C-H bonds is an essential reaction in biology and chemistry. Metalloenzymes that often exhibit this type of reactivity contain metal-oxido intermediates that are directly involved in the initial cleavage of the C-H bonds. Regulation of the cleavage process is achieved, in part, by hydrogen bonds that are proximal to the metal-oxido units, yet our understanding of their exact role(s) is still emerging. To gain further information into the role of H-bonds on C-H bond activation, a hybrid set of urea-containing tripodal ligands has been developed in which a single H-bond can be adjusted through changes in the properties of one ureayl N-H bond. This modularity is achieved by appending a phenyl ring with different para-substituents from one ureayl NH group. The ligands have been used to prepare a series of MnIII-oxido complexes, and a Hammett correlation was found between the pKa values of the complexes and the substituents on the phenyl ring that was explained within the context of changes to the H-bonds involving the MnIII-oxido unit. The complexes were tested for their reactivity toward 9,10-dihydroanthracene (DHA), and a Hammett correlation was found between the second-order rate constants for the reactions and the pKa values. Studies to determine activation parameters and the kinetic isotope effects are consistent with a mechanism in which rate-limiting proton transfer is an important contributor. However, additional reactivity studies with xanthene found a significant increase in the rate constant compared to DHA, even though the substrates have the same pKa(C-H) values. These results do not support a discrete proton-transfer/electron-transfer process, but rather an asynchronous mechanism in which the proton and electron are transferred unequally at the transition state.

Mechanisms of Alkyl and Aryl Thiol Addition to N-Methylmaleimide.

A mechanistic study was undertaken to elucidate the reaction pathways for thiol addition to N-methylmaleimide in water. We used linear free energy relationships, solvent kinetic isotope effects (SKIEs), activation parameters, and ionic strength effects to probe the nature of the rate-limiting transition states. Calculations were also employed and assisted in illuminating three possible mechanistic pathways: (1) stepwise addition with rate-limiting nucleophilic attack, (2) stepwise addition with rate-limiting proton transfer, and (3) concerted addition with nucleophilic attack and proton transfer occurring concurrently. Alkyl thiolate addition exhibits βnucRS-= 0.4, small negative Δ S‡ values, prominent ionic strength effects, and no evidence of general acid catalysis, consistent with pathway 1. Aryl thiolate addition exhibited βnucArS- = 1.0, large negative Δ S‡ values, normal primary SKIEs, general acid catalysis, and negligible sensitivity to ionic strength, consistent with pathways 2 and 3. The experimental and computational data depict an energy surface where ground state effects, namely the energy of the alkyl/aryl thiolate, play a major role in shaping the governing pathway. Application of these findings to bioconjugation chemistry is also discussed.

Ab Initio QM/MM Modeling of the Rate-Limiting Proton Transfer Step in the Deamination of Tryptamine by Aromatic Amine Dehydrogenase.

Aromatic amine dehydrogenase (AADH) and related enzymes are at the heart of debates on the roles of quantum tunneling and protein dynamics in catalysis. The reaction of tryptamine in AADH involves significant quantum tunneling in the rate-limiting proton transfer step, shown by large H/D primary kinetic isotope effects (KIEs), with unusual temperature dependence. We apply correlated ab initio combined quantum mechanics/molecular mechanics (QM/MM) methods, at levels up to local coupled cluster theory (LCCSD(T)/(aug)-cc-pVTZ), to calculate accurate potential energy surfaces for this reaction, which are necessary for quantitative analysis of tunneling contributions and reaction dynamics. Different levels of QM/MM treatment are tested. Multiple pathways are calculated with fully flexible transition state optimization by the climbing-image nudged elastic band method at the density functional QM/MM level. The average LCCSD(T) potential energy barriers to proton transfer are 16.7 and 14.0 kcal/mol for proton transfer to the two carboxylate atoms of the catalytic base, Asp128β. The results show that two similar, but distinct pathways are energetically accessible. These two pathways have different barriers, exothermicity and curvature, and should be considered in analyses of the temperature dependence of reaction and KIEs in AADH and other enzymes. These results provide a benchmark for this prototypical enzyme reaction and will be useful for developing empirical models, and analyzing experimental data, to distinguish between different conceptual models of enzyme catalysis.

Characterization of the Dynamic Equilibrium between Closed and Open Forms of the Benzoxaborole Pharmacophore.

Benzoxaboroles are a class of five-membered hemiboronic acids that recently attracted significant attention as a new pharmacophore on account of their unique structural and physicochemical properties and their ability to interact selectively with biomolecules. Their structural behavior in water and its effect on their physiological properties remain unclear, especially the question of dynamic hydrolytic equilibrium of the oxaborole ring. Herein, we used NMR spectroscopy, in mixed aqueous-organic solvent, to confirm the strong preference for the closed form of benzoxaborole and its six- and seven-membered homologues over the open boronic acid form. Only with the eight-membered homologue does the cyclic form become unfavorable. Using dynamic VT-NMR studies with designed probe compound 20, we demonstrate that the oxaborole ring undergoes rapid hydrolytic ring closing-opening at ambient temperature at a rate of >100 Hz via a mechanism featuring rate-limiting proton-transfer steps. This knowledge can help provide a better understanding of the behavior of benzoxaboroles in biological systems.

Concentration-dependent solvent effect on the SNAr reaction between 1-fluoro-2,4-dinitrobenzene and morpholine

The nature and extent of preferential solvation in SNAr reaction between 1-fluoro-2,4-dinitrobenzene and morpholine are observed to depend upon the concentration of amine. Positive deviation from ideality is observed during kinetic studies of reactions carried out with lower concentration of the amine, while reaction rates measured for systems containing higher concentration of the amine show negative deviation from ideal behavior. The anomaly originates from the competition between rate-limiting proton transfer and fluoride abstraction step in the SNAr mechanism. The observations have been explained on the basis of the generally accepted mechanism and by calculation of preferential solvation parameters.

Charge Tags for Most Comprehensive ESI-MS Monitoring of Morita-Baylis-Hillman (MBH)/aza-MBH Reactions: Solid Mechanistic View and the Dualistic Role of the Charge Tagged Acrylate.

Neutral and charge tagged reagents were used to investigate the mechanism of the classical Morita-Baylis-Hillman (MBH) reaction as well as its aza-version using mass spectrometry with electrospray ionization (ESI-MS). The use of an acrylate (activated alkene) with a methylimidazolium ion as a charge tag eliminates the requirement for adding acids as ESI(+) additives, which are normally used to favor protonation and therefore detection of reaction partners (reagents, intermediates, and products) by ESI(+)-MS. For both charge tagged reactions (MBH/aza-MBH), most reactants, intermediates, and the final adducts were efficiently detected in the form of abundant doubly and singly charged ions. Characterization of the reactions partners was performed via both tandem mass spectrometry (ESI(+)-MS/MS) and accurate m/z measurements. The charge tagged reactions also showed faster conversion rates when compare to the neutral reaction, indicating a dualistic role for the charge tagged acrylate. It acts as both the reagent and a cocatalyst due to the inherent ionic-coordination nature of the methylimidazolium ion, which stabilizes the zwitterionic intermediates and reagents through different types of coordination ion pairs. Hemiacetal intermediates for the rate-limiting proton transfer step were also intercepted and characterized for both classical and aza-MBH charge tagged reactions.

Binding small molecules and ions to [Fe4S4Cl4](2-) modulates rate of protonation of the cluster.

The mechanism of the acid-catalyzed substitution reaction of the terminal chloro-ligands in [Fe4S4Cl4](2-) by PhS(-) in the presence of NHBu(n)3(+) involves rate-limiting proton transfer from NHBu(n)3(+) to the cluster (k0 = 490 ± 20 dm(3) mol(-1) s(-1)). A variety of small molecules and ions (L = substrate = Cl(-), Br(-), I(-), RNHNH2 (R = Me or Ph), Me2NNH2, HCN, NCS(-), N3(-), Bu(t)NC or pyridine) bind to [Fe4S4Cl4](2-) and this affects the rate of subsequent protonation of [Fe4S4Cl4(L)](n-). Where the kinetics allow, the equilibrium constants for the substrates binding to [Fe4S4Cl4](2-) (K(L)) and the rates of proton transfer from NHBu(n)3(+) to [Fe4S4Cl4(L)](n-) (k) have been determined. The results indicate the following general features. (i) Bound substrates increase the rate of protonation of the cluster, but the rate increase is modest (k/k0 = 1.6 to ≥72). (ii) When K(L) is small, so is k/k0. (iii) Binding substrates which are good σ-donors or good π-acceptors lead to the largest k/k0. This behaviour is discussed in terms of the recent proposal that protonation of [Fe4S4Cl4](2-) at a μ3-S, is coupled to concomitant Fe-(μ3-SH) bond elongation/cleavage.

Cysteine Is the General Base That Serves in Catalysis by Isocitrate Lyase and in Mechanism-Based Inhibition by 3-Nitropropionate

Isocitrate lyase (ICL) catalyzes the reversible cleavage of isocitrate into succinate and glyoxylate. It is the first committed step in the glyoxylate cycle used by some organisms, including Mycobacterium tuberculosis, where it has been shown to be essential for cell survival during chronic infection. The pH-rate and pD-rate profiles measured in the direction of isocitrate synthesis revealed solvent kinetic isotope effects (KIEs) of 1.7 ± 0.4 for (D2O)V and 0.56 ± 0.07 for (D2O)(V/Ksuccinate). Whereas the (D2O)V is consistent with partially rate-limiting proton transfer during formation of the hydroxyl group of isocitrate, the large inverse (D2O)(V/Ksuccinate) indicates that substantially different kinetic parameters exist when the enzyme is saturated with succinate. Inhibition by 3-nitropropionate (3-NP), a succinate analogue, was found to proceed through an unusual double slow-onset process featuring formation of a complex with a Ki of 3.3 ± 0.2 μM during the first minute, followed by formation of a final complex with a Ki* of 44 ± 10 nM over the course of several minutes to hours. Stopped-flow measurements during the first minute revealed an apparent solvent KIE of 0.40 ± 0.03 for association and unity for dissociation. In contrast, itaconate, a succinate analogue lacking an acidic α-proton, did not display slow-binding behavior and yielded a (D2O)Ki of 1.0 ± 0.2. These results support a common mechanism for catalysis with succinate and inhibition by 3-NP featuring (1) an unfavorable prebinding isomerization of the active site Cys191-His193 pair to the thiolate-imidazolium form, a process that is favored in D2O, and (2) the transfer of a proton from succinate or 3-NP to Cys191. These findings also indicate that propionate-3-nitronate, which is the conjugate base of 3-NP and the "true inhibitor" of ICL, does not bind directly and must be generated enzymatically.

Redox-Linked Conformational Control of Proton-Coupled Electron Transfer: Y122 in the Ribonucleotide Reductase β2 Subunit

Tyrosyl radicals play essential roles in biological proton-coupled electron transfer (PCET) reactions. Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides and is vital in DNA replication in all organisms. Class Ia RNRs consist of α2 and β2 homodimeric subunits. In class Ia RNR, such as the E. coli enzyme, an essential tyrosyl radical (Y122O(•))-diferric cofactor is located in β2. Although Y122O(•) is extremely stable in free β2, Y122O(•) is highly reactive in the quaternary substrate-α2β2 complex and serves as a radical initiator in catalytic PCET between β2 and α2. In this report, we investigate the structural interactions that control the reactivity of Y122O(•) in a model system, isolated E. coli β2. Y122O(•) was reduced with hydroxyurea (HU), a radical scavenger that quenches the radical in a clinically relevant reaction. In the difference FT-IR spectrum, associated with this PCET reaction, amide I (CO) and amide II (CN/NH) bands were observed. Specific (13)C-labeling of the tyrosine C1 carbon assigned a component of these bands to the Y122-T123 amide bond. Comparison to density functional calculations on a model dipeptide, tyrosine-threonine, and structural modeling demonstrated that PCET is associated with a Y122 rotation and a 7.2 Å translation of the Y122 phenolic oxygen. To test for the functional consequences of this structural change, a proton inventory defined the origin of the large solvent isotope effect (SIE = 16.7 ± 1.0 at 25 °C) on this reaction. These data suggest that the one-electron, HU-mediated reduction of Y122O(•) is associated with two, rate-limiting (full or partial) proton transfer reactions. One is attributable to HU oxidation (SIE = 11.9, net H atom transfer), and the other is attributable to coupled, hydrogen-bonding changes in the Y122O(•)-diferric cofactor (SIE = 1.4). These results illustrate the importance of redox-linked changes to backbone and ring dihedral angles in high potential PCET and provide evidence for rate-limiting, redox-linked hydrogen-bonding interactions between Y122O(•) and the iron cluster.

A quantitative analysis of spontaneous isoaspartate formation from N-terminal asparaginyl and aspartyl residues

The formation of isoaspartate (isoAsp) from asparaginyl or aspartyl residues is a spontaneous post-translational modification of peptides and proteins. Due to isopeptide bond formation, the structure and possibly function of peptides and proteins is altered. IsoAsp modifications within the peptide chain have been reported for many cytosolic proteins. Amyloid peptides (Aβ) deposited in Alzheimer's disease may carry an N-terminal isoAsp-modification. Here, we describe a quantitative investigation of isoAsp-formation from N-terminal Asn and Asp using model peptides similar to the Aβ N-terminus. The study is based on a newly developed separation of peptides using capillary electrophoresis (CE). 1H NMR was employed to validate the basic finding of N-terminal isoAsp-formation from Asp and Asn. Thereby, the isomerization of Asn at neutral pH (0.6 day(-1), peptide NGEF) is approximately six times faster than that within the peptide chain (AANGEF). The difference in velocity between Asn and Asp isomerization is approximately 50-fold. In contrast to N-terminal Asn, Asp isomerization is significantly accelerated at acidic pH. The kinetic solvent isotope (kD2O/kH2O) effect of 2.46 suggests a rate-limiting proton transfer in isoAsp-formation. The proton inventory is consistent with transfer of one proton in the transition state, supporting the previous notion of rate-limiting deprotonation of the peptide backbone amide during succinimide-intermediate formation. The study provides evidence for a spontaneous N-terminal isoAsp-formation within peptides and might explain the accumulation of N-terminal isoAsp in amyloid deposits.

Kinetic and equilibrium studies of σ-adduct formation and nucleophilic substitution in the reactions of 2-chloro-3,5-dinitropyridine and 2-ethoxy-3,5-dinitropyridine with p-substituted anilines in DMSO

Rate and equilibrium results for the reactions of 2-chloro-3,5-dinitropyridine and 2-ethoxy-3,5-dinitropyridine with a series of p-substituted anilines in the presence of 1,4-diazabicyclo[2.2.2]octane (DABCO) were studied in DMSO. The reactions yielded 2-anilino-3,5-dinitropyridine derivatives, and no accumulation of intermediates could be detected spectrophotometrically. The rates were compatible with a two-step mechanism involving initial nucleophilic attack followed by either base-catalysed or uncatalysed conversion to the product. The base-catalysed pathway was likely to involve rate-limiting proton transfer from the zwitterionic intermediate to the base to yield the anionic σ-adduct. Plots of log K 1 k DABCO against pK a values gave good straight lines at 25 °C, with slopes of 0.42 for 2-chloro- and 0.45 for 2-ethoxy-3,5-dinitropyridine. The results were compared with those for the reactions of 2-phenoxy-3,5-dinitropyridine with substituted anilines.

Structural Water Cluster As a Possible Proton Acceptor in the Adduct Decay Reaction of Oat Phototropin 1 LOV2 Domain

LOV domains (Light, Oxygen, Voltage) are the light-sensory modules of phototropins, the blue-light photoreceptor kinases in plants, and of a wide variety of flavoproteins found in all three domains of life. These 12 kDa modules bind a flavin chromophore (FMN or FAD) noncovalently and undergo a photochemical activation in which the sulfur atom of a conserved cysteine forms an adduct to the C(4a) carbon of the flavin. The adduct breaks spontaneously in a base-catalyzed reaction involving a rate-limiting proton-transfer step, regenerating the dark state in seconds. This photocycle involves chromophore and protein structural changes that activate the C-terminal serine/threonine kinase. Previous studies (Biochemistry 2007, 46, 7016-7021) showed that decreased hydration obtained at high glycerol concentrations stabilizes the adduct state in a manner similar to that attained at low temperatures, resulting in much longer adduct decay times. This kinetic effect was attributed to an increased protein rigidity that hindered structural fluctuations necessary for the decay reaction. In this work, we studied the adduct decay kinetics of oat phototropin 1 (phot1) LOV2 at varying hydration using a specially designed chamber that allowed for measurement of UV-visible and FTIR spectra of the same samples. Therefore, we obtained LOV protein concentrations, adduct decay kinetics, and the different populations of bound water by deconvolution of the broad water absorption peak around 3500 cm(-1). A linear dependence of the adduct decay rate constant on the concentration of double and triple hydrogen-bonded waters strongly suggests that the adduct decay is a pseudo-first-order reaction in which both the adduct and the strongly bound waters are reactants. We suggest that a cluster of strongly bound water functions as the proton acceptor in the rate-limiting step of adduct decay.

Understanding the Role of Tyrosine 381 in the Activity of E. coli Aminopeptidase N (PepN)

Aminopeptidase N (PepN) from E. coli is an M1 class, Zn(II)‐dependent peptidase with broad specificity. PepN was mechanistically characterized in an effort to understand the role of tyrosine 381. pH Dependence kinetic studies revealed a log kcat versus pH and kcat/KM versus pH curves with inflection points at a pH of 9.0. Solvent isotope studies suggest at least one rate‐limiting proton transfer in the mechanism. In an effort to characterize the functional roles of binding region components, several active‐site amino acids were mutated. A tyrosine to alanine mutated PepN (Y381A) was successfully over‐expressed, purified, and characterized. Y381A‐PepN maintained Zn(II) binding efficiency but exhibited greater than 900‐fold loss of activity (kcat) when compared to the wild‐type PepN. This suggests that Tyr381 has an essential mechanistic role in the function of PepN beyond the suggested role of hydrogen bond stabilization of substrate and product. This work was funded by Augustana College New Faculty Research Fund, Augustana College Research Foundation, and Miami University.