The fragmentation-recombination mechanism of the enzyme glutamate mutase studied by QM/MM simulations

The radical mechanism of the conversion of glutamate to methylaspartate catalyzed by glutamate mutase is studied with quantum mechanical/molecular mechanical (QM/MM) simulations based on density functional theory (DFT/MM). The hydrogen transfer between the substrate and the cofactor is found to be rate limiting with a barrier of 101.1 kJ mol(-1). A careful comparison to the uncatalyzed reaction in water is performed. The protein influences the reaction predominantly electrostatically and to a lesser degree sterically. Our calculations shed light on the atomistic details of the reaction mechanism. The well-known arginine claw and Glu 171 ( Clostridium cochlearium notation) are found to have the strongest influence on the reaction. However, a catalytic role of Glu 214, Lys 322, Gln 147, Glu 330, Lys 326, and Met 294 is found as well. The arginine claw keeps the intermediates in place and is probably responsible for the enantioselectivity. Glu 171 temporarily accepts a proton from the glutamyl radical intermediate and donates it back at the end of the reaction. We relate our results to experimental data when available. Our simulations lead to further understanding of how glutamate mutase catalyzes the carbon skeleton rearrangement of glutamate.     

Toward an improved understanding of the glutamate mutase system

High-level quantum chemistry calculations have been used to examine the catalytic reactions of adenosylcobalamin-dependent glutamate mutase (GM) with the natural substrate (S)-glutamic acid. We have also examined the rearrangement of (S)-2-hydroxyglutaric acid, (S)-2-thiolglutaric acid, and 2-ketoglutaric acid, all of which have previously been shown to react as substrates or inhibitors of the enzyme. Our calculations support the notion that the 100-fold difference in kcat between glutamate and 2-hydroxyglutarate is associated with the relatively high energy of the glycolyl radical intermediate compared with the glycyl radical. More generally, calculations of radical stabilization energies for a variety of substituted glycyl radical analogues indicate that modifications at the radical center can profoundly affect the relative stability of the resulting radical, leading to important mechanistic consequences. We find that the formation of a thioglycolyl radical, derived from (S)-2-thiolglutaric acid, is highly dependent on the protonation state of sulfur. The neutral radical is found to be of stability similar to that of the glycolyl radical, whereas the S- form of the thioglycolyl radical is much more stable, thus providing a rationalization for the inhibition of the enzyme by the substrate analogue 2-thiolglutarate. Two possible rearrangement pathways have been examined for the reaction of GM with 2-ketoglutaric acid, for which previous experiments had suggested no rearrangement took place. The fragmentation-recombination pathway is associated with a fragmentation step that is very endothermic (by 102.2 kJ mol-1). In contrast, the addition-elimination pathway has significantly lower energy requirements. An alternative possibility, namely, that 2-ketoglutaric acid is bound in its hydrated form, 2,2-dihydroxyglutaric acid, also leads to a pathway with relatively low energy requirements, suggesting that some rearrangement might be expected under such circumstances.     

Computational mutation analysis of hydrogen abstraction and radical rearrangement steps in the catalysis of coenzyme B12-dependent diol dehydratase

A mutation analysis of the catalytic functions of active-site residues of coenzyme B(12)-dependent diol dehydratase in the conversion of 1,2-propanediol to 1,1-propanediol has been carried out by using QM/MM computations. Mutants His143Ala, Glu170Gln, Glu170Ala, and Glu170Ala/Glu221Ala were considered to estimate the impact of the mutations of His143 and Glu170. In the His143Ala mutant the activation energy for OH migration increased to 16.4 from 11.5 kcal mol(-1) in the wild-type enzyme. The highest activation energy, 19.6 kcal mol(-1), was measured for hydrogen back-abstraction in this reaction. The transition state for OH migration is not sufficiently stabilized by the hydrogen-bonding interaction formed between the spectator OH group and Gln170 in the Glu170Gln mutant, which demonstrates that a strong proton acceptor is required to promote OH migration. In the Glu170Ala mutant, a new strong hydrogen bond is formed between the spectator OH group and Glu221. A computed activation energy of 13.6 kcal mol(-1) for OH migration in the Glu170Ala mutant is only 2.1 kcal mol(-1) higher than the corresponding barrier in the wild-type enzyme. Despite the low activation barrier, the Glu170Ala mutant is inactive because the subsequent hydrogen back-abstraction is energetically demanding in this mutant. OH migration is not feasible in the Glu170Ala/Glu221Ala mutant because the activation barrier for OH migration is greatly increased by the loss of COO(-) groups near the spectator OH group. This result indicates that the effect of partial deprotonation of the spectator OH group is the most important factor in reducing the activation barrier for OH migration in the conversion of 1,2-propanediol to 1,1-propanediol catalyzed by diol dehydratase.     

Effect of side chains on competing pathways for beta-scission reactions of peptide-backbone alkoxyl radicals

High-level quantum chemistry calculations have been carried out to investigate beta-scission reactions of alkoxyl radicals located at the alpha-carbon of a peptide backbone. This type of alkoxyl radical may undergo three possible beta-scission reactions, namely C-C beta-scission of the backbone, C-N beta-scission of the backbone, and C-R beta-scission of the side chain. We find that the rates for the C-C beta-scission reactions are all very fast, with rate constants of the order 10(12) s(-1) that are essentially independent of the side chain. The C-N beta-scission reactions are all slow, with rate constants that range from 10(-0.7) to 10(-4.5) s(-1). The rates of the C-R beta-scission reactions depend on the side chain and range from moderately fast (10(7) s(-1)) to very fast (10(12) s(-1)). The rates of the C-R beta-scission reactions correlate well with the relative stabilities of the resultant side-chain product radicals (*R), as reflected in calculated radical stabilization energies (RSEs). The order of stabilities for the side-chain fragment radicals for the natural amino acids is found to be Ala < Glu < Gln approximately Leu approximately Met approximately Lys approximately Arg < Asp approximately Ile approximately Asn approximately Val < Ser approximately Thr approximately Cys < Phe approximately Tyr approximately His approximately Trp. We predict that for side-chain C-R beta-scission reactions to effectively compete with the backbone C-C beta-scission reactions, the side-chain fragment radicals would generally need an RSE greater than approximately 30 kJ mol(-1). Thus, the residues that may lead to competitive side-chain beta-scission reactions are Ser, Thr, Cys, Phe, Tyr, His, and Trp.     

Interconversion of (S)-glutamate and (2S,3S)-3-methylaspartate: a distinctive B(12)-dependent carbon-skeleton rearrangement

The interconversion of (S)-glutamate and (2S,3S)-3-methylaspartate catalyzed by B(12)-dependent glutamate mutase is discussed using results from high-level ab initio molecular orbital calculations. Evidence is presented regarding the possible role of coenzyme-B(12) in substrate activation and product formation via radical generation. Calculated electron paramagnetic resonance parameters support experimental evidence for the involvement of substrate-derived radicals and will hopefully aid the future detection of other important radical intermediates. The height of the rearrangement barrier for a fragmentation-recombination pathway, calculated with a model that includes neutral amino and carboxylic acid substituents in the migrating glycyl group, supports recent experimental evidence for the interconversion of (S)-glutamate and (2S,3S)-3-methylaspartate through such a pathway. Our calculations suggest that the enzyme may facilitate the rearrangement of (S)-glutamate through (partial) proton-transfer processes that control the protonation state of substituents in the migrating group.     

Enzymatic radical catalysis: coenzyme B12-dependent diol dehydratase

A peculiar function resides in a peculiar structure. Coenzyme B₁₂ or adenosylcobalamin, a naturally occurring organometallic compound, serves as a cofactor for enzymatic radical reactions. How do the enzymes form catalytic radicals at the active sites? How do the enzymes utilize and control the high reactivity of the radicals for catalysis? Recently, three‐dimensional structures of several radical‐containing or radical‐forming enzymes including B₁₂ enzymes have been reported, enabling the analysis of the fine mechanisms of the action of these interesting enzymes. Our biochemical, mutational, and crystallographic studies as well as theoretical calculations on diol dehydratase, an adenosylcobalamin–dependent enzyme, revealed that its structure is adapted for its function—that is, activation of the Co? C bond toward homolysis, abstraction of a specific hydrogen atom from the substrate and its recombination to a particular product, and transition state stabilization in the hydroxyl group migration of a substrate‐derived radical. The functions of K⁺ and the active‐site amino acid residues in enzyme catalysis are also investigated. Based on the results, the fine mechanism of the enzyme and the energetic feasibility of enzymatic radical catalysis are described here. © 2002 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 2: 352–366, 2002: Published online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.10035     

Time-resolved measurements of the photolysis and recombination of adenosylcobalamin bound to glutamate mutase

Femtosecond to nanosecond transient absorption spectroscopy is used to investigate the photolysis of 5'-deoxyadenosylcobalamin (coenzyme B12, AdoCbl) bound to glutamate mutase. The photochemistry of AdoCbl is found to be inherently dependent upon the environment of the cofactor. Excitation of AdoCbl bound to glutamate mutase results in formation of a metal-to-ligand charge transfer intermediate state which decays to form cob(II)alamin with a time constant of 105 ps. This observation is in contrast to earlier measurements in water where the photohomolysis proceeds through an intermediate state in which the axial dimethylbenzimidazole ligand appears to have dissociated, and measurements in ethylene glycol where prompt bond homolysis is observed (Yoder, L. M.; Cole, A. G.; Walker, L. A., II; Sension, R. J. J. Phys. Chem. B 2001, 105, 12180-12188). The quantum yield for formation of stable radical pairs in the enzyme is found to be phi = 0.05 +/- 0.03, and the resulting intrinsic rate constants for geminate recombination and "cage escape" are 1.0 +/- 0.1 and 0.05 +/- 0.03 ns(-1), respectively. The rate constant for geminate recombination is 30% less than that observed for AdoCbl in water or ethylene glycol. This reduction is insufficient to account for the 10(12)-fold increase in the homolysis rate observed when substrate is bound to the protein. Finally, the protein provides a cage to prevent diffusive loss of the adenosyl radical; however, the ultimate yield for long-lived radicals is determined by the evolution from a singlet to a triplet radical pair as proposed for AdoCbl in ethylene glycol.     

Protein engineering of nitrile hydratase activity of papain: molecular dynamics study of a mutant and wild-type enzyme

The mechanism of hydrolysis of the nitrile (N-acetyl-phenylalanyl-2-amino-propionitrile, I) catalyzed by Gln19Glu mutant of papain has been studied by nanosecond molecular dynamics (MD) simulations. MD simulations of the complex of mutant enzyme with I and of mutant enzyme covalently attached to both neutral (II) and protonated (III) thioimidate intermediates were performed. An MD simulation with the wild-type enzyme.I complex was undertaken as a reference. The ion pair between protonated His159 and thiolate of Cys25 is coplanar, and the hydrogen bonding interaction S(-)(25).HD1-ND1(159) is observed throughout MD simulation of the mutant enzyme.I complex. Such a sustained hydrogen bond is absent in nitrile-bound wild-type papain due to the flexibility of the imidazole ring of His159. The nature of the residue at position 19 plays a critical role in the hydrolysis of the covalent thioimidate intermediate. When position 19 represents Glu, the imidazolium ion of His159-ND1(+).Cys25-S(-) ion pair is distant, on average, from the nitrile nitrogen of substrate I. Near attack conformers (NACs) have been identified in which His159-ImH(+) is positioned to initiate a general acid-catalyzed addition of Cys-S(-) to nitrile. Though Glu19-CO(2)H is distant from nitrile nitrogen in the mutant.I structure, MD simulations of the mutant.II covalent adduct finds Glu19-CO(2)H hydrogen bonded to the thioimide nitrogen of II. This hydrogen bonded species is much less stable than the hydrogen bonded Glu19-CO(2)(-) with mutant-bound protonated thioimidate (III). This observation supports Glu19-CO(2)H general acid catalysis of the formation of mutant.III. This is the commitment step in the Gln19Glu mutant catalysis of nitrile hydrolysis.     

Enzymatic coupling of specific peptides at nonspecific ligation sites: effect of Asp189Glu mutation in trypsin on substrate mimetic-mediated reactions

Two main drawbacks seriously restrict the synthetic value of proteases as reagents in peptide fragment coupling: (i) native proteolytic activity and, thus, risk of undesired peptide cleavage; (ii) limited enzyme specificities restricting the amino acid residues between which a peptide bond can be formed. While the latter can be overcome by the use of substrate mimetics achieving peptide bond formation at nonspecific ligation sites, the risk of proteolytic cleavage still remains and hinders the wide acceptance of this powerful strategy for peptide coupling. This paper reports on the effect of the trypsin point mutant Asp189Glu on substrate mimetic-mediated reactions. The effect of this mutation on the steady-state hydrolysis of substrate mimetics of the 4-guanidinophenyl ester type and on trypsin-specific Lys- and Arg-containing peptides was investigated. The results were confirmed by enzymatic coupling reactions using substrate mimetics as the acyl donor and specific amino acid-containing peptides as the acyl acceptor. The competition assay verifies the predicted shift in substrate preference from Lys and Arg to the substrate mimetics and, thus, from cleavage to synthesis of peptide bonds. The combination of results obtained qualifies the trypsin mutant D189E as the first substrate mimetic-specific peptide ligase.     

Deamidation and transamidation of substance P by tissue transglutaminase revealed by electron-capture dissociation fourier transform mass spectrometry

Tissue transglutaminase (tTGase) catalyzes both deamidation and transamidation of peptides and proteins by using a peptidyl glutamine as primary substrate. A precise consensus sequence for the enzyme is unknown and the ratio between deamidated and transamidated (or cross‐linked) reaction products is highly substrate‐dependent. Due to its overlapping body distribution with tTGase and ease of manipulation with tandem mass spectrometry, we used the neuropeptide substance P as a model to investigate the associated enzymatic kinetics and reaction products. Online liquid‐chromatography Fourier‐transform ion‐cyclotron‐resonance mass spectrometry (FT‐ICR MS) combined with electron‐capture dissociation (ECD) was employed to study the tTGase‐induced modifications of substance P. A particular strength of ECD for peptide‐enzyme reaction product monitoring is its ability to distinguish isomeric amino acids, for example, Glu and iso‐Glu, by signature product ions. Our studies show that the primary reaction observed is deamidation, with the two consecutive glutamine residues converted sequentially into glutamate: first Gln₅, and subsequently Gln₆. We then applied ECD FT‐ICR MS to identify the transamidation site on an enzymatically cross‐linked peptide, which turned out to correspond to Gln₅. Three populations of substance‐P dimers were detected that differed by the number of deamidated Gln residues. The higher reactivity of Gln₅ over Gln₆ was further confirmed by cross‐linking SP with monodansylcadaverine (MDC). Overall, our approach described herein is of a general importance for mapping both enzymatically induced post‐translational protein modifications and cross‐linking. Finally, in vitro Ca‐signaling assays revealed that the main tTGase reaction product, the singly deamidated SP (RPKPEQFFGLM‐NH₂), has increased agonist potency towards its natural receptor, thus confirming the biologically relevant role of deamidation.     

Stabilisation of methylene radicals by cob(II)alamin in coenzyme B12 dependent mutases

Coenzyme B12 initiates radical chemistry in two types of enzymatic reactions, the irreversible eliminases (e.g., diol dehydratases) and the reversible mutases (e.g., methylmalonyl-CoA mutase). Whereas eliminases that use radical generators other than coenzyme B12 are known, no alternative coenzyme B12 independent mutases have been detected for substrates in which a methyl group is reversibly converted to a methylene radical. We predict that such mutases do not exist. However, coenzyme B12 independent pathways have been detected that circumvent the need for glutamate, beta-lysine or methylmalonyl-CoA mutases by proceeding via different intermediates. In humans the methylcitrate cycle, which is ostensibly an alternative to the coenzyme B12 dependent methylmalonyl-CoA pathway for propionate oxidation, is not used because it would interfere with the Krebs cycle and thereby compromise the high-energy requirement of the nervous system. In the diol dehydratases the 5'-deoxyadenosyl radical generated by homolysis of the carbon-cobalt bond of coenzyme B12 moves about 10 A away from the cobalt atom in cob(II)alamin. The substrate and product radicals are generated at a similar distance from cob(II)alamin, which acts solely as spectator of the catalysis. In glutamate and methylmalonyl-CoA mutases the 5'-deoxyadenosyl radical remains within 3-4 A of the cobalt atom, with the substrate and product radicals approximately 3 A further away. It is suggested that cob(II)alamin acts as a conductor by stabilising both the 5'-deoxyadenosyl radical and the product-related methylene radicals.     

How an enzyme tames reactive intermediates: positioning of the active-site components of lysine 2,3-aminomutase during enzymatic turnover as determined by ENDOR spectroscopy

Lysine 2,3-aminomutase (LAM) utilizes a [4Fe-4S] cluster, S-adenosyl-L-methionine (SAM), and pyridoxal 5'-phosphate (PLP) to isomerize L-alpha-lysine to L-beta-lysine. LAM is a member of the radical-SAM enzyme superfamily in which a [4Fe-4S]+ cluster reductively cleaves SAM to produce the 5'-deoxyadenosyl radical, which abstracts an H-atom from substrate to form 5'-deoxyadenosine (5'-Ado) and the alpha-Lys* radical (state 3 (Lys*)). This radical isomerizes to the beta-Lys* radical (state 4(Lys*)), which then abstracts an H-atom from 5'-Ado to form beta-lysine and the 5'-deoxyadenosyl radical; the latter then regenerates SAM. We use 13C, 1,2H, 31P, and 14N ENDOR to characterize the active site of LAM in intermediate states that contain the isomeric substrate radicals or analogues. With L-alpha-lysine as substrate, we monitor the state with beta-Lys*. In parallel, we use two substrate analogues that generate stable analogues of the alpha-Lys* radical: trans-4,5-dehydro-L-lysine (DHLys) and 4-thia-L-lysine (SLys). This first glimpse of the motions of active-site components during catalytic turnover suggests a possible major movement of PLP during catalysis. However, the principal focus of this work is on the relative positions of the carbons involved in H-atom transfer. We conclude that the active site facilitates hydrogen atom transfer by enforcing van der Waals contact between radicals and their reacting partners. This constraint enables the enzyme to minimize and even eliminate side reactions of highly reactive species such as the 5'-deoxyadensosyl radical.     

Spectroscopic and kinetic studies of PKU-inducing mutants of phenylalanine hydroxylase: Arg158Gln and Glu280Lys

Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin-dependent, nonheme iron enzyme that catalyzes the hydroxylation of L-Phe to L-Tyr in the rate-limiting step of phenylalanine catabolism. This reaction is tightly coupled in the wild-type enzyme to oxidation of the tetrahydropterin cofactor. Dysfunction of PAH activity in humans leads to the disease phenylketonuria (PKU). We have investigated two PKU-inducing mutants, Arg158Gln and Glu280Lys, using kinetic methods, magnetic circular dichrosim (MCD) spectroscopy, and X-ray absorption spectroscopy (XAS). Analysis of the products produced by the mutant enzymes shows that although both oxidize pterin at more than twice the rate of wild-type enzyme, these reactions are only approximately 20% coupled to production of L-Tyr. Previous MCD and XAS studies had demonstrated that the resting Fe(II) site is six-coordinate in the wild-type enzyme and converts to a five-coordinate site when both L-Phe and reduced pterin are present in the active site. Although the Arg158Gln mutant forms the five-coordinate site when both cosubstrates are bound, the Fe(II) site of the Glu280Lys mutant remains six-coordinate. These results provide insight into the PAH reaction and disease mechanism at a molecular level, indicating that the first step of the mechanism is formation of a peroxy-pterin species, which subsequently reacts with the Fe(II) site if the pterin is properly oriented for formation of an Fe-OO-pterin bridge and an open coordination position is available on the Fe(II).     

Quantum Mechanics/Molecular Mechanics Studies on the Mechanism of Action of Cofactor Pyridoxal 5′‐Phosphate in Ornithine 4,5‐Aminomutase

A computational study was performed on the experimentally elusive cyclisation step in the cofactor pyridoxal 5′‐phosphate (PLP)‐dependent D ‐ornithine 4,5‐aminomutase (OAM)‐catalysed reaction. Calculations using both model systems and a combined quantum mechanics/molecular mechanics approach suggest that regulation of the cyclic radical intermediate is achieved through the synergy of the intrinsic catalytic power of cofactor PLP and the active site of the enzyme. The captodative effect of PLP is balanced by an enzyme active site that controls the deprotonation of both the pyridine nitrogen atom (N1) and the Schiff‐base nitrogen atom (N2). Furthermore, electrostatic interactions between the terminal carboxylate and amino groups of the substrate and Arg297 and Glu81 impose substantial “strain” energy on the orientation of the cyclic intermediate to control its trajectory. In addition the “strain” energy, which appears to be sensitive to both the number of carbon atoms in the substrate/analogue and the position of the radical intermediates, may play a key role in controlling the transition of the enzyme from the closed to the open state. Our results provide new insights into several aspects of the radical mechanism in aminomutase catalysis and broaden our understanding of cofactor PLP‐dependent reactions.     

A meanfield approach to the thermodynamics of a protein-solvent system with application to the oligomerization of the tumor suppressor p53

The thermodynamic stability and oligomerization status of the tumor suppressor p53 tetramerization domain have been studied experimentally and theoretically. A series of hydrophilic mutations at Met-340 and Leu-344 of human p53 were designed to disrupt the hydrophobic dimer-dimer interface of the tetrameric oligomerization domain of p53. Meanfield calculations of the free energy of the solvated mutants as a function of interdimer distance were compared with experimental data on the thermal stability and oligomeric state [tetramer, dimer, or equilibrium mixture of both] of each mutant. The calculations predicted a decreasing stability and oligomeric state for the following amino acids at residue 340: Met [tetramer] > Ser Asp, His, Gin, > Glu, Lys [dimer], whereas the experimental results showed the following order: Met [tetramer] > Ser > Gln > His, Lys > Asp, Glu [dimers]. For residue 344, the calculated trend was Leu [tetramer] > Ala > Arg, Gln, Lys [dimer], and the experimental trend was Leu [tetramer] > Ala, Arg, Gln, Lys [dimer]. The discrepancy for the lysine side chain at residue 340 is attributed to the dual nature of lysine, both hydrophobic and charged. The incorrect prediction of stability of the mutant with Asp at residue 340 is attributed to the fact that within the meanfield approach, we use the wild-type backbone configuration for all mutants, but low melting temperatures suggest a softening of the α-helices at the dimer-dimer interface. This initial application of meanfield theory toward a protein-solvent system is encouraging for the application of the theoretical model to more complex systems.     

Stabilisation of Methylene Radicals by Cob(II)alamin in Coenzyme B12 Dependent Mutases

Coenzyme B₁₂ initiates radical chemistry in two types of enzymatic reactions, the irreversible eliminases (e.g., diol dehydratases) and the reversible mutases (e.g., methylmalonyl‐CoA mutase). Whereas eliminases that use radical generators other than coenzyme B₁₂ are known, no alternative coenzyme B₁₂ independent mutases have been detected for substrates in which a methyl group is reversibly converted to a methylene radical. We predict that such mutases do not exist. However, coenzyme B₁₂ independent pathways have been detected that circumvent the need for glutamate, β‐lysine or methylmalonyl‐CoA mutases by proceeding via different intermediates. In humans the methylcitrate cycle, which is ostensibly an alternative to the coenzyme B₁₂ dependent methylmalonyl‐CoA pathway for propionate oxidation, is not used because it would interfere with the Krebs cycle and thereby compromise the high‐energy requirement of the nervous system. In the diol dehydratases the 5′‐deoxyadenosyl radical generated by homolysis of the carbon–cobalt bond of coenzyme B₁₂ moves about 10 Å away from the cobalt atom in cob(II )alamin. The substrate and product radicals are generated at a similar distance from cob(II )alamin, which acts solely as spectator of the catalysis. In glutamate and methylmalonyl‐CoA mutases the 5′‐deoxyadenosyl radical remains within 3–4 Å of the cobalt atom, with the substrate and product radicals approximately 3 Å further away. It is suggested that cob(II )alamin acts as a conductor by stabilising both the 5′‐deoxyadenosyl radical and the product‐related methylene radicals.     

Monitoring the Dynamics of Monomer Exchange Using Electrospray Mass Spectrometry: The Case of the Dimeric Glucosamine-6-Phosphate Synthase

Escherichia coli glucosamine-6-phosphate synthase (GlmS) is a dimeric enzyme from the glutamine-dependent amidotransferases family, which catalyses the conversion of D-fructose-6-phosphate (Fru6P) and glutamine (Gln) into D-glucosamine-6-phosphate (GlcN6P) and glutamate, respectively. Extensive X-ray crystallography investigations have been reported, highlighting the importance of the dimeric association to form the sugar active site as well as significant conformational changes of the protein upon substrate and product binding. In the present work, an approach based on time-resolved noncovalent mass spectrometry has been developed to study the dynamics of GlmS subunit exchange. Using ¹⁴N versus ¹⁵N labeled proteins, the kinetics of GlmS subunit exchange was monitored with the wild-type enzyme in the presence of different substrates and products as well as with the protein bearing a key amino acid mutation specially designed to weaken the dimer interface. Determination of rate constants of subunit exchange revealed important modifications of the protein dynamics: while glutamine, glutamate, and K603A mutation accelerates subunit exchange, Fru6P and GlcN6P totally prevent it. These results are described in light of the available structural information, providing additional useful data for both the characterization of GlmS catalytic process and the design of new GlmS inhibitors. Finally, time-resolved noncovalent MS can be proposed as an additional biophysical technique for real-time monitoring of protein dynamics.     

THE PHOTOREDUCTION OF FLAVINS BY AMINO ACIDS AND EDTA. A CONTINUOUS AND FLASH PHOTOLYSIS STUDY

Abstract— Primary and secondary photochemical processes in oxygen-free aqueous solution have been characterised for FMN alone and in the presence of EDTA and four amino acids using nanosecond and microsecond flash photolysis and continuous photolysis techniques. The relative contributions of oneelectron and two-electron (group or hydride transfer) reactions to the deactivation of the triplet has been determined by comparing the radical concentration (560 nm) with the bleaching of the ground state (446 nm). It was concluded that one-electron reactions (hydrogen atom or electron abstraction) are the major mode of reactivity of the flavin triplet state with all the suhstrates studied.
The nature of the reactions of the flavin semiquinone radical have been studied quantitatively by microsecond flash photolysis. These secondary reactions consist of either a 'back reaction' between the flavin and substrate radicals (tryptophan or glycyl-tyrosine) or the transfer of a second electron (or hydrogen atom) from the substrate radical to the flavin radical (EDTA, methionine and possibly cysteine) to form reduced flavin and oxidised substrate. From a comparison of the quantum yields of formation of reduced flavin using 'flash' and continuous irradiation, an additional pathway for the decay of the flavin radical is suggested to occur at low light intensities in the presence of glycyl-tyrosine or histidine.     

Pulse radiolysis studies of short-lived species in solid amino acids as precursors of radicals and detected by ESR

The aim of the study was to bring closer solid state radiation chemistry and ESR spectroscopy by looking for precursors of free radicals which give ESR signals. It has been performed using time-resolved spectrophotometry (pulse radiolysis of the solid state) and diffuse reflection spectrophotometry. Alanine has been especially considered as the most investigated amino acid, important for radiation dosimetry. Absorption of the transient (Λ maximum at 460 nm) is identified as the species during deamination. The stable absorption spectrum with the Λ maximum at 345 nm is due to the same radical as the one detected by ESR. Other amino acids: valine, threonine, glutamine and arginine show similar behaviour: microsecond spectrum of the intermediate appears always at longer wavelenghts. The transient spectrum changes into stable absorption in UV of a lower wavelenght. Along with the optical spectrum, the ESR spectrum appears, of similar stability. Also, other features indicate that the same radical is responsible for both the electronic and ESR spectrum. Some amino acids, like methionine give intensive transient absorption in the microsecond range but no ESR signal, after completion of consecutive fast reactions. In that case any optical absorption is due to the stable product of radiolysis, i.e. compounds with paired electrons only.     

The efficiency of immobilised glutamate oxidase decreases with surface enzyme loading: an electrostatic effect, and reversal by a polycation significantly enhances biosensor sensitivity

The apparent Michaelis constant, K(M), for glutamate oxidase (GluOx) immobilised on Pt electrodes increased systematically with enzyme loading. The effect was due, at least in part, to electrostatic repulsion between neighbouring oxidase molecules and the anionic substrate, glutamate (Glu). This understanding has allowed us to increase the Glu sensitivity of GluOx-based amperometric biosensors in the linear response region (100+/-11 nA cm(-2)microM(-1) at pH 7.4; SD, n=23) by incorporating a polycation (polyethyleneimine, PEI) to counterbalance the polyanionic protein. Differences in the behaviour of glucose biosensors of a similar configuration highlight a limitation of using glucose oxidase as a model enzyme in biosensor design.