The influence of aspartate 26 on the tautomeric forms of folate bound to Lactobacillus casei dihydrofolate reductase.

The ternary complex of Lactobacillus casei dihydrofolate reductase (DHFR) with folate and NADP+ exists as a mixture of three interconverting forms (I, IIa and IIb) whose relative populations are pH dependent, with an effective pK of approx. 6. To investigate the role of Asp26 in this pH dependence we have measured the 13C chemical shifts of [2,4a,7,9-(13)C4]folate in its complex with the mutant DHFR Asp26 --> Asn and NADP+. Only a single form of the complex is detected and this has the characteristics of form I, an enol form with its N1 unprotonated. A study of the pH dependence of the 13C chemical shifts of DHFR selectively labelled with [4-(13)C]aspartic acid in its complex with folate and NADP+ indicates that no Asp residue has a pK value greater than 5.4. Two of the Asp CO2 signals appear as non-integral signals with chemical shifts typical of non-ionised COOH groups and with a pH dependence characteristic of the slow exchange equilibria previously characterised for signals in forms I and IIb (or IIa). It is proposed that the protonation/deprotonation controlling the equilibria involves the O4 position of the folate and that Asp26 influences this indirectly by binding in its CO2 form to the protonated N1 group of folate in forms I and IIa thus reducing the pK involving protonation at the O4 position to approx. 6. These findings indicate that, in forms I and IIa of the ternary complex, folate binds to DHFR in a very similar way to methotrexate.

Data acquisition system using six degree-of-freedom inertia sensor and ZigBee wireless link for fall detection and prevention.

Fall detection and prevention require logged physiological activity data of a patient for a long period of time. This work develops a data acquisition system to collect motion data from multiple patients and store in a data base. A wireless sensor network is built using high precision inertia sensors and low power Zigbee wireless transceivers. Testing results prove the system function properly. Researchers and physicians can now retrieve and analyze the accurate data of the patient movement with ease.

An impulse based sensor for medical sensing applications.

This work designs a non-coherent impulse basedtransceiver operating in a frequency range of 3.1-10.6 GHz for medical sensing applications. The transmitter consists of an ON/OFF Keying data modulator, a Gaussian pulse generator, and a variable gain amplifier to control the transmitting pulse level. The receiver consists of an LNA, a multiplier, an integrator, and a comparator. The IC is designed using 0.18 microm CMOS technology with a supply voltage of 1.8 V. The simulated pulse width is 0.2 ns and the maximum pulse rate is over 1 GHz. A heart motion detection performance was demonstrated with high precision for an overall power consumption of 40 mW. This design can also be modified to be used in wireless UWB data communications to build a complete low power wireless sensor node.

Flavoenzyme catalysed oxidation of amines: roles for flavin and protein-based radicals.

Amines are a carbon source for the growth of a number of bacterial species and they also play key roles in neurotransmission, cell growth and differentiation, and neoplastic cell proliferation. Enzymes have evolved to catalyse these reactions and these oxidoreductases can be grouped into the flavoprotein and quinoprotein families. The mechanism of amine oxidation catalysed by the quinoprotein amine oxidases is understood reasonably well and occurs through the formation of enzyme-substrate covalent adducts with TPQ (topaquinone), TTQ (tryptophan tryptophylquinone), CTQ (cysteine tryptophylquinone) and LTQ (lysine tyrosyl quinone) redox centres. Oxidation of amines by flavoenzymes is less well understood. The role of protein-based radicals and flavin semiquinone radicals in the oxidation of amines is discussed.

New insights into enzyme catalysis. Ground state tunnelling driven by protein dynamics.

The wave-particle duality of matter suggests that quantum tunnelling may have a prominent role in enzymatic H-transfer. However, unlike for electron tunnelling, evidence for H-tunnelling in enzyme molecules is extremely limited. The theoretical development, and verification by experiment, of a role for protein dynamics in driving enzymatic H-tunnelling is presented. Dynamic theories of H-tunnelling suggest that the kinetic isotope effect, during rupture of a C-H/C-D bond, for example, can assume values interpreted previously as indicating classical transfer. Vibrationally enhanced ground state tunnelling has been demonstrated for enzymes that cleave stable C-H bonds. This is an attractive mechanism as large activation energies make it energetically unfavourable for a classical, over-the-barrier mode of cleavage. Furthermore, it may be a general strategy used by enzymes for catalysing these 'difficult' transformations.

Enzymatic H-transfer requires vibration-driven extreme tunneling.

Enzymatic breakage of the substrate C-H bond by Methylophilus methyltrophus (sp. W3A1) methylamine dehydrogenase (MADH) has been studied by stopped-flow spectroscopy. The rate of reduction of the tryptophan tryptophylquinone (TTQ) cofactor has a large kinetic isotope effect (KIE = 16.8 +/- 0.5), and the KIE is independent of temperature. Analysis of the temperature dependence of C-H bond breakage revealed that extreme (ground state) quantum tunneling is responsible for the transfer of the hydrogen nucleus. Reaction rates are strongly dependent on temperature, indicating thermally induced, vibrational motion drives the H-transfer reaction. The data provide direct experimental evidence for enzymatic bond breakage by extreme tunneling driven by vibrational motion of the protein scaffold. The results demonstrate that classical transition state theory and its tunneling derivatives do not adequately describe this enzymatic reaction.

Optimizing the Michaelis complex of trimethylamine dehydrogenase: identification of interactions that perturb the ionization of substrate and facilitate catalysis with trimethylamine base.

Recent evidence from isotope studies supports the view that catalysis by trimethylamine dehydrogenase (TMADH) proceeds from a Michaelis complex involving trimethylamine base and not, as thought previously, trimethylammonium cation. In native TMADH reduction of the flavin by substrate (perdeuterated trimethylamine) is influenced by two ionizations in the Michaelis complex with pK(a) values of 6.5 and 8.4; maximal activity is realized in the alkaline region. The latter ionization has been attributed to residue His-172 and, more recently, the former to the ionization of substrate itself. In the Michaelis complex, the ionization of substrate (pK(a) approximately 6.5 for perdeuterated substrate) is perturbed by approximately -3.3 to -3.6 pH units compared with that of free trimethylamine (pK(a) = 9.8) and free perdeuterated trimethylamine (pK(a) = 10.1), respectively, thus stabilizing trimethylamine base by approximately 2 kJ mol(-1). We show, by targeted mutagenesis and stopped-flow studies that this reduction of the pK(a) is a consequence of electronic interaction with residues Tyr-60 and His-172, thus these two residues are key for optimizing catalysis in the physiological pH range. We also show that residue Tyr-174, the remaining ionizable group in the active site that we have not targeted previously by mutagenesis, is not implicated in the pH dependence of flavin reduction. Formation of a Michaelis complex with trimethylamine base is consistent with a mechanism of amine oxidation that we advanced in our previous computational and kinetic studies which involves nucleophilic attack by the substrate nitrogen atom on the electrophilic C4a atom of the flavin isoalloxazine ring. Stabilization of trimethylamine base in the Michaelis complex over that in free solution is key to optimizing catalysis at physiological pH in TMADH, and may be of general importance in the mechanism of other amine dehydrogenases that require the unprotonated form of the substrate for catalysis.

Deuterium isotope effects during carbon-hydrogen bond cleavage by trimethylamine dehydrogenase. Implications for mechanism and vibrationally assisted hydrogen tunneling in wild-type and mutant enzymes.

His-172 and Tyr-169 are components of a triad in the active site of trimethylamine dehydrogenase (TMADH) comprising Asp-267, His-172, and Tyr-169. Stopped-flow kinetic studies with trimethylamine as substrate have indicated that mutation of His-172 to Gln reduces the limiting rate constant for flavin reduction approximately 10-fold (Basran, J., Sutcliffe, M. J., Hille, R., and Scrutton, N. S. (1999) Biochem. J. 341, 307-314). A kinetic isotope effect (KIE = k(H)/k(D)) accompanies flavin reduction by H172Q TMADH, the magnitude of which varies significantly with solution pH. With trimethylamine, flavin reduction by H172Q TMADH is controlled by a single macroscopic ionization (pK(a) = 6.8 +/- 0.1). This ionization is perturbed (pK(a) = 7.4 +/- 0.1) in reactions with perdeuterated trimethylamine and is responsible for the apparent variation in the KIE with solution pH. At pH 9.5, where the functional group controlling flavin reduction is fully ionized, the KIE is independent of temperature in the range 277-297 K, consistent with vibrationally assisted hydrogen tunneling during breakage of the substrate C-H bond. Y169F TMADH is approximately 4-fold more compromised than H172Q TMADH for hydrogen transfer, which occurs non-classically. Studies with Y169F TMADH suggest partial thermal excitation of substrate prior to hydrogen tunneling by a vibrationally assisted mechanism. Our studies illustrate the varied effects of compromising mutations on tunneling regimes in enzyme molecules.

Electron transfer from flavin to iron in the Pseudomonas oleovorans rubredoxin reductase-rubredoxin electron transfer complex.

Rubredoxin reductase (RR) and rubredoxin form a soluble and physiological eT complex. The complex provides reducing equivalents for a membrane-bound omega-hydroxylase, required for the hydroxylation of alkanes and related compounds. The gene (alkT) encoding RR has been overexpressed and the enzyme purified in amounts suitable for studies of eT by stopped-flow spectroscopy. The eT reactions from NADH to the flavin of RR and from reduced RR to the 1Fe and 2Fe forms of rubredoxin have been characterized by transient kinetic and thermodynamic analysis. The reductive half-reaction proceeds in a one-step reaction involving oxidized enzyme and a two-electron-reduced enzyme-NAD+ charge-transfer complex. Flavin reduction is observed at 450 nm and charge-transfer formation at 750 nm; both steps are hyperbolically dependent on NADH concentration. The limiting flavin reduction rate (180 +/- 4 s-1) is comparable to the limiting rate for charge-transfer formation (189 +/- 7 s-1) and analysis at 450 and 750 nm yielded enzyme-NADH dissociation constants of 36 +/- 2 and 43 +/- 5 microM, respectively. Thermodynamic analysis of the reductive half-reaction yielded values for changes in entropy (DeltaS = -65.8 +/- 2.2 J mol-1 K-1), enthalpy (DeltaH = 37.8 +/- 0.6 kJ mol-1) and Gibbs free energy (DeltaG = 57.5 +/- 0.7 kJ mol-1 at 298 K) during hydride ion transfer to the flavin N5 atom. Spectral analysis of mixtures of 1Fe or 2Fe rubredoxin and RR suggest that conformational changes accompany eT complex assembly. Both the 1Fe (nonphysiological) and 2Fe (physiological) forms of rubredoxin were found to oxidize two electron-reduced rubredoxin reductase with approximately equal facility. Rates for the reduction of rubredoxin are hyperbolically dependent on rubredoxin concentration and the limiting rates are 72. 7 +/- 0.6 and 55.2 +/- 0.3 s-1 for the 1Fe and 2Fe forms, respectively. Analysis of the temperature dependence of eT to rubredoxin using eT theory revealed that the reaction is not adequately described as a nonadiabatic eT reaction (HAB >> 80 cm-1). eT to both the 1Fe and 2Fe forms of rubredoxin is therefore gated by an adiabatic process that precedes the eT reaction from flavin to iron. Possible origins of this adiabatic event are discussed.

X-ray scattering studies of Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein. Evidence for multiple conformational states and an induced fit mechanism for assembly with trimethylamine dehydrogenase.

Small angle x-ray solution scattering has been used to generate a low resolution, model-independent molecular envelope structure for electron-transferring flavoprotein (ETF) from Methylophilus methylotrophus (sp. W(3)A(1)). Analysis of both the oxidized and 1-electron-reduced (anionic flavin semiquinone) forms of the protein revealed that the solution structures of the protein are similar in both oxidation states. Comparison of the molecular envelope of ETF from the x-ray scattering data with previously determined structural models of the protein suggests that ETF samples a range of conformations in solution. These conformations correspond to a rotation of domain II with respect to domains I and III about two flexible "hinge" sequences that are unique to M. methylotrophus ETF. The x-ray scattering data are consistent with previous models concerning the interaction of M. methylotrophus ETF with its physiological redox partner, trimethylamine dehydrogenase. Our data reveal that an "induced fit" mechanism accounts for the assembly of the trimethylamine dehydrogenase-ETF electron transfer complex, consistent with spectroscopic and modeling studies of the assembly process.

High-level expression and isotopic labeling of Lactobacillus casei dihydrofolate reductase for nuclear magnetic resonance spectroscopy.

Two efficient systems have been used for high-level expression of Lactobacillus casei dihydrofolate reductase in Escherichia coli, including the production of protein generally and specifically labeled with 13C and 15N. A system based on T7 RNA polymerase led to the production of dihydrofolate reductase at a level of 37% of the total soluble protein of the host strain: 50 mg of pure enzyme was obtained from a 1 liter of culture (or 14 mg/g wet weight of cells). In this system, a small amount of the enzyme (less than 5%) was identified as a catalytically active 21-kDa fusion protein. Introduction of a second in-frame (ochre) stop codon did not eliminate the production of this fusion protein. The same expression system was also used to prepare dihydrofolate reductase generally labeled with 15N and to prepare single and double mutants of the enzyme. In order to have an expression system which can be used with a range of auxotrophic strains of E. coli, a system based on the tac promoter was used. This led to the production of dihydrofolate reductase at a level of 29% of total soluble protein; a yield of 40 mg enzyme per liter of culture (or 11 mg/g wet weight of cells). This system was successfully used to produce mutants of the enzyme as well as the enzyme selectively labeled with [gamma-13C]aspartic acid.

Role of the active-site carboxylate in dihydrofolate reductase: kinetic and spectroscopic studies of the aspartate 26-->asparagine mutant of the Lactobacillus casei enzyme.

A mutant of Lactobacillus casei dihydrofolate reductase, D26N, in which the active site aspartic acid residue has been replaced by asparagine by oligonucleotide-directed mutagenesis has been studied by NMR and optical spectroscopy and its kinetic behavior characterized in detail. On the basis of comparisons of a large number of chemical shifts and NOEs, it is clear that there are only very slight structural differences between the methotrexate complexes of the wild-type and mutant enzymes and that these are restricted to the immediate environment of the substitution. The data suggest a slight difference in orientation of the pteridine ring in the binding site in the mutant enzyme. Both NMR and UV spectroscopy show that methotrexate is protonated on N1 when bound to the wild-type enzyme but not when bound to the mutant. Binding constant measurements by fluorescence quenching and steady-state kinetic measurements of dihydrofolate (FH2) and folate reduction show that the substitution has little or no effect on substrate, coenzyme, and inhibitor binding (< 7-fold increase in Kd) and only a modest effect on kcat (up to a factor of 9 for FH2 and 25 for folate) and kcat/KM (up to a factor of 13 for FH2 and 14 for folate). Measurements of deuterium isotope effects and direct measurements of hydride ion transfer and product release by stopped-flow methods revealed that for the mutant enzyme hydride ion transfer is rate-limiting across the pH range 5-8. This allowed a direct comparison of the rate of hydride ion transfer in the wild-type and mutant enzymes; the asparagine substitution was found to decrease this rate by 62-fold at pH 5.5 and 9-fold at pH 7.5. This effect is much smaller than that seen for the corresponding mutant of Escherichia coli dihydrofolate reductase [Howell, E. E., Villafranca, J. E., Warren, M. S., Oatley, S. J., & Kraut, J. (1986) Science 231, 1123-1128], estimated as a 1000-fold decrease in the rate of hydride ion transfer. The change in pH dependence of kcat resulting from the substitution is consistent with, but does not prove, the idea that the group of pK 6.0 which must be protonated for hydride ion transfer to occur is Asp26. For folate reduction, the pH dependence of kcat is determined by two pKs, one of which, pK 5, disappears in the mutant enzyme, suggesting that it may correspond to ionization of Asp26.(ABSTRACT TRUNCATED AT 400 WORDS)

Involvement of a flavin iminoquinone methide in the formation of 6-hydroxyflavin mononucleotide in trimethylamine dehydrogenase: a rationale for the existence of 8alpha-methyl and C6-linked covalent flavoproteins.

In trimethylamine dehydrogenase, substrate is bound in the active site via cation-pi bonding to three aromatic residues (Tyr-60, Trp-264, and Trp-355). Mutation of one of these residues (Trp-355 --> Leu, mutant W355L) influences the chemistry of the flavin mononucleotide in the active site, enabling derivatization to 6-hydroxy-FMN. The W355L mutant is purified as a mixture of deflavo, natural 6-S-cysteinyl-FMN, and inactive 6-hydroxy-FMN forms, and the enzyme is severely compromised in its ability to oxidatively demethylate trimethylamine. Analysis of samples of the native and recombinant wild-type trimethylamine dehydrogenases also revealed the presence of 6-hydroxy-FMN, but at much reduced levels compared with that of the W355L enzyme. Unlike that for a C30A mutant of trimethylamine dehydrogenase, addition of substrate to the W355L trimethylamine dehydrogenase is not required for the production of 6-hydroxy-FMN. A mechanism is proposed for the 6-hydroxylation of FMN in trimethylamine dehydrogenase that involves an electrophilic flavin iminoquinone methide. The proposed mechanism involving the flavin iminoquinone methide could apply to the flavinylation of trimethylamine dehydrogenase at the C6 position but also to the flavinylation of enzymes via the 8alpha position, thus providing a rationale for the evolution of covalent flavoproteins in general. Covalent linkage at C6 or the 8alpha-methyl prevents 6-hydroxylation by direct modification at the C6 atom or by preventing formation of the flavin iminoquinone methide, respectively.

The reaction of trimethylamine dehydrogenase with trimethylamine.

The reductive half-reaction of trimethylamine dehydrogenase with its physiological substrate trimethylamine has been examined by stopped-flow spectroscopy over the pH range 6.0-11.0, with attention focusing on the fastest of the three kinetic phases of the reaction, the flavin reduction/substrate oxidation process. As in previous work with the slow substrate diethylmethylamine, the reaction is found to consist of three well resolved kinetic phases. The observed rate constant for the fast phase exhibits hyperbolic dependence on the substrate concentration with an extrapolated limiting rate constant (klim) greater than 1000 s-1 at pH above 8.5, 10 degrees C. The kinetic parameter klim/Kd for the fast phase exhibits a bell-shaped pH dependence, with two pKa values of 9.3 +/- 0.1 and 10. 0 +/- 0.1 attributed to a basic residue in the enzyme active site and the ionization of the free substrate, respectively. The sigmoidal pH profile for klim gives a single pKa value of 7.1 +/- 0. 2. The observed rate constants for both the intermediate and slow phases are found to decrease as the substrate concentration is increased. The steady-state kinetic behavior of trimethylamine dehydrogenase with trimethylamine has also been examined, and is found to be adequately described without invoking a second, inhibitory substrate-binding site. The present results demonstrate that: (a) substrate must be protonated in order to bind to the enzyme; (b) an ionization group on the enzyme is involved in substrate binding; (c) an active site general base is involved, but not strictly required, in the oxidation of substrate; (d) the fast phase of the reaction with native enzyme is considerably faster than observed with enzyme isolated from Methylophilus methylotrophus that has been grown up on dimethylamine; and (e) a discrete inhibitory substrate-binding site is not required to account for excess substrate inhibition, the kinetic behavior of trimethylamine dehydrogenase can be readily explained in the context of the known properties of the enzyme.

The role of Tyr-169 of trimethylamine dehydrogenase in substrate oxidation and magnetic interaction between FMN cofactor and the 4Fe/4S center.

Tyr-169 in trimethylamine dehydrogenase is one component of a triad also comprising residues His-172 and Asp-267. Its role in catalysis and in mediating the magnetic interaction between FMN cofactor and the 4Fe/4S center have been investigated by stopped-flow and EPR spectroscopy of a Tyr-169 to Phe (Y169F) mutant of the enzyme. Tyr-169 is shown to play an important role in catalysis (mutation to phenylalanine reduces the limiting rate constant for bleaching of the active site flavin by about 100-fold) but does not serve as a general base in the course of catalysis. In addition, we are able to resolve two kinetically influential ionizations involved in both the reaction of free enzyme with free substrate (as reflected in klim/Kd), and in the breakdown of the Eox.S complex (as reflected in klim). In EPR studies of the Y169F mutant, it is found that the ability of the Y169F enzyme to form the spin-interacting state between flavin semiquinone and reduced 4Fe/4S center characteristic of wild-type enzyme is significantly compromised. The present results are consistent with Tyr-169 representing the ionizable group of pKa approximately 9.5, previously identified in pH-jump studies of electron transfer, whose deprotonation must occur for the spin-interacting state to be established.

Effects of single-residue substitutions on negative cooperativity in ligand binding to dihydrofolate reductase.

The effects of six amino acid substitutions in Lactobacillus casei dihydrofolate reductase, predominantly in the coenzyme binding site, on catalysis and on the negative cooperativity between NADPH and tetrahydrofolate binding have been determined. Replacement of Leu62, His64 or Leu54 by alanine has no effect on kcat, and produces only modest changes in negative cooperativity. Alanine substitution of His77, which interacts indirectly with the coenzyme adenine ring, leads to a doubling of the negative cooperativity and a consequent doubling of kcat. Replacement of Arg43, which interacts with the coenzyme 2'-phosphate, by alanine, or of Trp21, which interacts with the coenzyme nicotinamide ring, by histidine leads to a 20-100-fold decrease in negative cooperativity. In both mutants there is a decrease in kcat; isotope effects show that product release is largely rate-limiting in R43A, whereas in W21H hydride ion transfer is rate-limiting. 1H NMR has been used to obtain information on the extent of the structural changes produced by the substitutions. This varies from very local effects in H64A to very widespread effects in W21H. These changes are used as the basis for discussion of the mechanisms of the functional effects of the various substitutions. It is suggested that residues in helix C, beta-strand 3 and the beta3-beta4 loop may be involved in the transmission of effects between the coenzyme and substrate binding sites.

Organization of the genes involved in dimethylglycine and sarcosine degradation in Arthrobacter spp.: implications for glycine betaine catabolism.

The nucleotide sequences of two cloned DNA fragments containing the structural genes of heterotetrameric sarcosine oxidase (soxBDAG) and dimethylglycine dehydrogenase (dmg) from Arthrobater spp. 1-IN and Arthrobacter globiformis, respectively, have been determined. Open reading frames were identified in the soxBDAG operon corresponding to the four subunits of heterotetrameric sarcosine oxidase by comparison with the N-terminal amino-acid sequences and the subunit relative molecular masses of the purified enzyme. Alignment of the deduced sarcosine oxidase amino-acid sequence with amino-acid sequences of functionally related proteins indicated that the arthrobacterial enzyme is highly homologous to sarcosine oxidase from Corynebacterium P-1. Deletion and expression analysis, and alignment of the deduced amino-acid sequence of the dmg gene, showed that dmg encodes a novel dimethylglycine oxidase, which is related to eukaryotic dimethylglycine dehydrogenase, and contains nucleotide-binding, flavinylation and folate-binding motifs. The recombinant dimethylglycine oxidase was purified to homogeneity and characterized. The DNA located upstream and downstream of both the soxBDAG and dmg genes is predicted to encode enzymes involved in the tetrahydrofolate-dependent assimilation of methyl groups. Based on the sequence analysis reported herein, pathways are proposed for glycine betaine catabolism in Arthrobacter species, which involve the identified folate-dependent enzymes.

Selective modification of alkylammonium ion specificity in trimethylamine dehydrogenase by the rational engineering of cation-pi bonding.

In trimethylamine dehydrogenase (TMADH), substrate is bound in the active site by organic cation-pi bonding mediated by residues Tyr-60, Trp-264, and Trp-355. In the closely related dimethylamine dehydrogenase (DMADH), modeling suggests that a mixture of cation-pi bonding and conventional hydrogen bonding is responsible for binding dimethylamine. The active sites of both enzymes are highly conserved, but three changes in amino acid identity (residues Tyr-60 --> Gln, Ser-74 --> Thr, and Trp-105 --> Phe, TMADH numbering) were identified as probable determinants for tertiary --> secondary alkylammonium ion specificity. In an attempt to switch the substrate specificity of TMADH so that the enzyme operates more efficiently with dimethylamine, three mutant proteins of TMADH were isolated. The mutant forms contained either a single mutation (Y60Q), double mutation (Y60Q x S74T) or triple mutation (Y60Q x S74T x W105F). A kinetic analysis in the steady state with trimethylamine and dimethylamine as substrate indicated that the specificity of the triple mutant was switched approximately 90,000-fold in favor of dimethylamine. The major component of this switch in specificity is a selective impairment of the catalytic efficiency of the enzyme with trimethylamine. Rapid-scanning and single wavelength stopped-flow spectroscopic studies revealed that the major effects of the mutations are on the rate of flavin reduction and the dissociation constant for substrate when trimethylamine is used as substrate. With dimethylamine as substrate, the rate constants for flavin reduction and the dissociation constants for substrate are not substantially affected in the mutant enzymes compared with wild-type TMADH. The results indicate a selective modification of the substrate-binding site in TMADH (that impairs catalysis with trimethylamine but not with dimethylamine) is responsible for the switch in substrate specificity displayed by the mutant enzymes.

Reductive half-reaction of the H172Q mutant of trimethylamine dehydrogenase: evidence against a carbanion mechanism and assignment of kinetically influential ionizations in the enzyme-substrate complex.

The reactions of wild-type trimethylamine dehydrogenase (TMADH) and of a His-172-->Gln (H172Q) mutant were studied by rapid-mixing stopped-flow spectroscopy over the pH range 6.0-10.5, to address the potential role of His-172 in abstracting a proton from the substrate in a 'carbanion' mechanism for C-H bond cleavage. The pH-dependence of the limiting rate for flavin reduction (klim) was studied as a function of pH for the wild-type enzyme with perdeuterated trimethylamine as substrate. The use of perdeuterated trimethylamine facilitated the unequivocal identification of two kinetically influential ionizations in the enzyme-substrate complex, with macroscopic pKa values of 6.5+/-0.2 and 8.4+/-0.1. A plot of klim/Kd revealed a bell-shaped curve and two kinetically influential ionizations with macroscopic pKa values of 9.4+/-0.1 and 10.5+/-0.1. Mutagenesis of His-172, a potential active-site base and a component of a novel Tyr-His-Asp triad in the active site of TMADH, revealed that the pKa of 8.4+/-0.1 for the wild-type enzyme-substrate complex represents ionization of the imidazolium side-chain of His-172. H172Q TMADH retains catalytic competence throughout the pH range investigated. At pH 10.5, and in contrast with the wild-type enzyme, flavin reduction in H172Q TMADH is biphasic. The fast phase is dependent on the trimethylamine concentration and exhibits a kinetic isotope effect of about 3; C-H bond cleavage is thus partially rate-limiting. In contrast, the slow phase does not show hyperbolic dependence on substrate concentration, and the observed rate shows no dependence on isotope, revealing that C-H bond cleavage is not rate-limiting. The analysis of H172Q TMADH, together with data recently acquired for the Y169F mutant of TMADH, reveals that C-H bond breakage is not initiated via abstraction of a proton from the substrate by an active-site base. The transfer of reducing equivalents to flavin via a carbanion mechanism is therefore unlikely.