Travels with carbon-centered radicals. 5'-deoxyadenosine and 5'-deoxyadenosine-5'-yl in radical enzymology.

As a graduate student under Professor R. H. Abeles, I began my journey with 5'-deoxyadenosine, studying the coenzyme B12 (adenosylcobalamin)-dependent dioldehydrase (DDH). I proved that suicide inactivation of dioldehydrase by glycolaldehyde proceeded with irreversible cleavage of adenosylcobalamin to 5'-deoxyadenosine. I further showed that suicide inactivation by [2-(3)H]glycolaldehyde produced 5'-deoxy[(3)H]adenosine, the first demonstration of hydrogen transfer to adenosyl-C5' of adenosylcobalamin. The tritium kinetic isotope effect (T)k was 15, which correlated well with the measurement (D)k = 12 for transformation of [1-(2)H]propane-1,2-diol to [2-(2)H]propionaldehyde by DDH. After establishing my own research program, I returned to the glycolaldehyde inactivation of DDH, showing by EPR that suicide inactivation produced glycolaldehyde-2-yl. In retrospect, suicide inactivation involved scission of adenosylcobalamin to 5'-deoxyadenosine-5'-yl, which abstracted a hydrogen from glycolaldehyde. Captodative-stabilized glycolaldehyde-2-yl could not react further, leading to suicide inactivation. In 1986, my colleagues and I took up the problem of the mechanism by which lysine 2,3-aminomutase (LAM) catalyzes S-adenosylmethionine (SAM) and pyridoxal-5'-phosphate (PLP)-dependent interconversion of l-lysine and l-ß-lysine. Because the reaction followed the pattern of adenosylcobalamin-dependent rearrangements, I postulated that SAM might be an evolutionary predecessor to adenosylcobalamin. Testing this hypothesis, we traced hydrogen transfer from lysine through the adenosyl-C5' of SAM to ß-lysine. Thus, the 5'-deoxyadenosyl of SAM mediated hydrogen transfer by LAM exactly as in adenosylcobalamin mediated hydrogen transfer in B12-dependent isomerizations. The mechanism postulated that SAM cleaves to form 5'-deoxyadenosine-5'-yl followed by abstraction of C3(H) from PLP-α-lysine aldimine to form PLP-α-lysine-3-yl. PLP-α-lysine-3-yl isomerizes to pyridoxal-ß-lysine-2-yl, and a hydrogen abstraction from 5'-deoxyadenosine regenerates 5'-deoxyadenosine-5'-yl and releases ß-lysine. Of four radicals in the postulated mechanism, three have been characterized by EPR spectroscopy as kinetically competent intermediates. The analysis of the role of iron allowed researchers to elucidate the mechanism by which SAM is cleaved to 5'-deoxyadenosine-5'-yl. LAM contains one [4Fe-4S] cluster ligated by three cysteine residues. As shown by ENDOR spectroscopy and X-ray crystallography, the fourth ligand to the cluster is SAM, through the methionyl carboxylate and amino groups. Inner sphere electron transfer within the [4Fe-4S](1+)-SAM complex leads to [4Fe-4S](2+)-Met and 5'-deoxyadenosine-5'-yl. The iron-binding motif in LAM, CxxxCxxC, found by other groups in four other SAM-dependent enzymes, is the founding motif for the radical SAM superfamily. These enzymes number in the tens of thousands and are responsible for highly diverse and chemically difficult transformations in the biosphere. Available information supports the hypothesis that this superfamily provides the chemical context from which the much more structurally complex adenosylcobalamin evolved.

Chemical and stereochemical actions of UDP-galactose 4-epimerase.

Uridine(5')diphospho(1)α-D-galactose (UDP-gal) provides all galactosyl units in biologically synthesized carbohydrates. All healthy cells produce UDP-gal from uridine(5')diphospho(1)α-D-glucose (UDP-glc) by the action of UDP-galactose 4-epimerase (GalE). This Account provides our recent results describing unusual mechanistic features of this enzyme. Fully active GalE is dimeric and contains one tightly bound nicotinamide adenine dinucleotide (NAD) per subunit. The NAD undergoes reversible reduction to NADH in the chemical mechanism. GalE displays unusual enzymological, chemical, and stereochemical properties. These include practically irreversible binding of NAD, nonstereospecific hydride transfer, uridine nucleotide-induced activation of NAD, Tyr149 as a base catalyst, and [GalE-NADH]-oxidation in one-electron steps by one-electron acceptors. Early studies revealed that uridine(5')diphospho(1)α-D-4-ketopyranose (UDP-4-ketopyranose) and NADH are reaction intermediates. Weak binding of the 4-ketopyranosyl moiety and strong binding of the UDP-moiety allowed either face of the 4-ketopyranosyl moiety to accept hydride from NADH. In crystal structures of GalE, NAD bound within a Rossmann-type fold and uridine nucleotides within a substrate domain. Structures of [GalE-NADH] in complex with UDP-glc show Lys153, Tyr149, and Ser124 in contact with NAD or glucosyl-C4(OH). Lys153 forms hydrogen bonds to the ribosyl-OH groups of NAD. The phenolate of Tyr149 is associated with both the nicotinamide ring of NAD and glucosyl-C4(OH). Ser124 is hydrogen-bonded to glucosyl-C4(OH). Spectrophotometry studies show a pH-dependent charge transfer (CT) complex between Tyr149 and NAD. The CT-complex has a pKa of 6.1, which results in bleaching of the CT-band. The CT-band also bleaches upon binding of a uridine nucleotide. Kinetic experiments with wild-type GalE and Ser124Ala-GalE show the same kinetic pKa values as the corresponding CT-band pKa, which point to Tyr149 as the base catalyst for hydride transfer. We used NMR studies to verify that uridine nucleotide binding polarizes nicotinamide π-electrons. The binding of uridine(5')-diphosphate (UDP) to GalE-[nicotinamide-1-¹5N]NAD shifts the ¹5N-signal upfield 3 ppm, whereas UDP-binding to GalE-[nicotinamide-4-¹³C]NAD shifts the ¹³C-signal downfield by 3.4 ppm. Electrochemical and ¹³C NMR data for a series of N-alkylnicotinamides show that the 3.4 ppm downfield ¹³C-perturbation in GalE corresponds to an elevation of the NAD reduction potential by 150 mV. These results account for the uridine nucleotide-dependence in the reduction of [GalE-NAD] by glucose or NaBH3CN. Kinetics in the reduction of Tyr149Phe- and Lys153Met-GalE-NAD implicate Tyr149 and Lys153 in the nucleotide-dependent reduction of NAD. They further implicate electrostatic repulsion between N1 of NAD and the ε-aminium group of Lys153 in nucleotide-induced activation of NAD. In an O2-dependent reaction, [GalE-NADH] reduces the stable radical UDP-TEMPO with production of superoxide radical. The reaction must proceed by way of a NAD-pyridinyl radical intermediate.

Mechanism-based inhibition reveals transitions between two conformational states in the action of lysine 5,6-aminomutase: a combination of electron paramagnetic resonance spectroscopy, electron nuclear double resonance spectroscopy, and density functional theory study.

An "open"-state crystal structure of lysine 5,6-aminomutase suggests that transition to a hypothetical "closed"-state is required to bring the cofactors adenosylcobalamin (AdoCbl) and pyridoxal-5'-phosphate (PLP) and the substrate into proximity for the radical-mediated 1,2-amino group migration. This process is achieved by transaldimination of the PLP-Lys144ß internal aldimine with the PLP-substrate external aldimine. A closed-state crystal structure is not available. UV-vis and electron paramagnetic resonance studies show that homologues of substrate D-lysine, 2,5-DAPn, 2,4-DAB, and 2,3-DAPr bind to PLP as an external aldimine and elicit the AdoCbl Co-C bond homolysis and the accumulations of cob(II)alamin and analogue-based radicals, demonstrating the existence of a closed state. (2)H- and (31)P-electron nuclear double resonance studies, supported by computations, show that the position for hydrogen atom abstraction from 2,5-DAPn and 2,4-DAB by the 5'-deoxyadenosyl radical occurs at the carbon adjacent to the imine, resulting in overstabilized radicals by spin delocalization through the imine into the pyridine ring of PLP. These radicals block the active site, inhibit the enzyme, and poise the enzyme into two distinct conformations: for even-numbered analogues, the cob(II)alamin remains proximal to and spin-coupled with the analogue-based radical in the closed state while odd-numbered analogues could trigger the transition to the open state of the enzyme. We provide here direct spectroscopic evidence that strongly support the existence of a closed state and its analogue-dependent transition to the open state, which is one step that was proposed to complete the catalytic turnover of the substrate lysine.

Pyridoxal-5'-phosphate as the catalyst for radical isomerization in reactions of PLP-dependent aminomutases.

PLP catalyzes the 1,2 shifts of amino groups in free radical-intermediates at the active sites of amino acid aminomutases. Free radical forms of the substrates are created upon H atom abstractions carried out by the 5'-deoxyadenosyl radical. In most of these enzymes, the 5'-deoxyadenosyl radical is generated by an iron-sulfur cluster-mediated reductive cleavage of S-adenosyl-(S)-methionine. However, in lysine 5,6-aminomutase and ornithine 4,5-aminomutase, the radical is generated by homolytic cleavage of the cobalt-carbon bond of adenosylcobalamin. The imine linkages in the initial radical forms of the external aldimines undergo radical addition to form azacyclopropylcarbinyl radicals as central intermediates in the catalytic cycles. In the case of lysine 2,3-aminomutase, the multistep catalytic mechanism is corroborated by direct spectroscopic observation and characterization of a product radical trapped during steady-state turnover. Analogues of the substrate-related radical having substituents adjacent to the radical center to stabilize the unpaired electron are also observed and characterized spectroscopically. A functional allylic analogue of the 5'-deoxyadenosyl radical is observed spectroscopically. A high-resolution crystal structure fully supports the mechanistic proposals. Evidence for a similar free radical mediated amino group transfer in the adenosylcobalamin-dependent lysine 5,6-aminomutase is provided by spectroscopic detection and characterization of radicals generated from the 4-thia analogues of the lysine substrates. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.

S-adenosylmethionine as an oxidant: the radical SAM superfamily.

A recently discovered superfamily of enzymes function using chemically novel mechanisms, in which S-adenosylmethionine (SAM) serves as an oxidizing agent in DNA repair and the biosynthesis of vitamins, coenzymes and antibiotics. Members of this superfamily, the radical SAM enzymes, are related by the cysteine motif CxxxCxxC, which nucleates the [4Fe-4S] cluster found in each. A common thread in the novel chemistry of these proteins is the use of a strong reducing agent--a low-potential [4Fe-4S](1+) cluster--to generate a powerful oxidizing agent, the 5'-deoxyadenosyl radical, from SAM. Recent results are beginning to determine the unique biochemistry for some of the radical SAM enzymes, for example, lysine 2,3 aminomutase, pyruvate formate lyase activase and biotin synthase.

Radical stabilization is crucial in the mechanism of action of lysine 5,6-aminomutase: role of tyrosine-263α as revealed by electron paramagnetic resonance spectroscopy.

Adenosylcobalamin- and pyridoxal-5'-phosphate-dependent lysine 5,6-aminomutase utilizes free radical intermediates to mediate 1,2-amino group rearrangement, during which an elusive high-energy aziridincarbinyl radical is proposed to be central in the mechanism of action. Understanding how the enzyme participates in stabilizing any of the radical intermediates is fundamentally significant. Y263F mutation abolished the enzymatic activity. With isotope-edited EPR methods, the roles of the Tyr263α residue in the putative active site are revealed. The Tyr263α residue stabilizes a radical intermediate, which most likely is the aziridincarbinyl radical, either by acting as a spin-relay device or serving as an anchor for the pyridine ring of pyridoxal-5'-phosphate through aromatic π-stacking interactions during spin transfer. The Tyr263α residue also protects the radical intermediate from interception by molecular oxygen. This study supports the proposed reaction mechanism, including the aziridincarbinyl radical, which has eluded detection for more than two decades.

The Radical SAM Superfamily.

The radical S-adenosylmethionine (SAM) superfamily currently comprises more than 2800 proteins with the amino acid sequence motif CxxxCxxC unaccompanied by a fourth conserved cysteine. The charcteristic three-cysteine motif nucleates a [4Fe-4S] cluster, which binds SAM as a ligand to the unique Fe not ligated to a cysteine residue. The members participate in more than 40 distinct biochemical transformations, and most members have not been biochemically characterized. A handful of the members of this superfamily have been purified and at least partially characterized. Significant mechanistic and structural information is available for lysine 2,3-aminomutase, pyruvate formate-lyase, coproporphyrinogen III oxidase, and MoaA required for molybdopterin biosynthesis. Biochemical information is available for spore photoproduct lyase, anaerobic ribonucleotide reductase activation subunit, lipoyl synthase, and MiaB involved in methylthiolation of isopentenyladenine-37 in tRNA. The radical SAM enzymes biochemically characterized to date have in common the cleavage of the [4Fe-4S](1 +) -SAM complex to [4Fe-4S](2 +)-Met and the 5' -deoxyadenosyl radical, which abstracts a hydrogen atom from the substrate to initiate a radical mechanism.

Radical triplets and suicide inhibition in reactions of 4-thia-D- and 4-thia-L-lysine with lysine 5,6-aminomutase.

Lysine 5,6-aminomutase (5,6-LAM) catalyzes the interconversions of D- or L-lysine and the corresponding enantiomers of 2,5-diaminohexanoate, as well as the interconversion of L-beta-lysine and l-3,5-diaminohexanoate. The reactions of 5,6-LAM are 5'-deoxyadenosylcobalamin- and pyridoxal-5'-phosphate (PLP)-dependent. Similar to other 5'-deoxyadenosylcobalamin-dependent enzymes, 5,6-LAM is thought to function by a radical mechanism. No free radicals can be detected by electron paramagnetic resonance (EPR) spectroscopy in reactions of 5,6-LAM with D- or L-lysine or with L-beta-lysine. However, the substrate analogues 4-thia-L-lysine and 4-thia-D-lysine undergo early steps in the mechanism to form two radical species that are readily detected by EPR spectroscopy. Cob(II)alamin and 5'-deoxyadenosine derived from 5'-deoxyadenosylcobalamin are also detected. The radicals are proximal to and spin-coupled with low-spin Co(2+) in cob(II)alamin and appear as radical triplets. The radicals are reversibly formed but do not proceed to stable products, so that 4-thia-D- and L-lysine are suicide inhibitors. Inhibition attains equilibrium between the active Michaelis complex and the inhibited radical triplets. The structure of the transient 4-thia-L-lysine radical is analogous to that of the first substrate-related radical in the putative isomerization mechanism. The second, persistent radical is more stable than the transient species and is assigned as a tautomer, in which a C6(H) of the transient radical is transferred to the carboxaldehyde carbon (C4') of PLP. The persistent radical blocks the active site and inhibits the enzyme, but it decomposes very slowly at

Evidence for conformational movement and radical mechanism in the reaction of 4-thia-L-lysine with lysine 5,6-aminomutase.

We demonstrate that the steady state reaction of lysine 5,6-aminomutase with substrate analogue 4-thia-l-lysine generates a radical intermediate, which accumulates in the enzyme to an electron paramagnetic resonance (EPR) detectable level. EPR line width narrowing of approximately 1 mT due to [4'-(2)H] labeling of the pyridoxal-5'-phosphate (PLP), an isotropic hyperfine coupling of 40 MHz for the proton at C4' of PLP derived from (2)H electron nuclear double resonance (ENDOR) measurement, and spin density delocalization onto the (31)P of PLP realized from observations of the (31)P ENDOR signal provide unequivocal identification of the radical as a substrate-PLP-based species. X- and Q-band EPR spectra fittings demonstrate that this radical is spin coupled with the low spin Co(2+) in cob (II) alamin and the distance between the two species is about 10 A. These results provide direct evidence for the active site motion upon substrate binding, bringing the adenosylcobalamin to the proximity of substrate-PLP for subsequent H-atom abstraction and for the notion that lysine 5,6-aminomutase functions by a radical mechanism. Observation of (2)H-ENDOR signal also provides a reliable hyperfine coupling constant for future comparison with quantum-mechanical-based calculations to gain further insight into the molecular structure of this steady state radical intermediate.

Glutamate 2,3-aminomutase: a new member of the radical SAM superfamily of enzymes.

A gene eam in Clostridium difficile encodes a protein that is homologous to lysine 2,3-aminomutase (LAM) in many other species but does not have the lysyl-binding residues Asp293 and Asp330 in LAM from Clostridium subterminale SB4. The C. difficile protein has Lys and Asn, respectively, in the sequence positions of the essential Asp residues in LAM. The C. difficile gene has been cloned into an E. coli expression vector, expressed in E. coli, and the protein purified and characterized. The recombinant protein displays excellent activity as a glutamate 2,3-aminomutase and no activity toward l-lysine. The PLP-, iron-, and sulfide-content and ultraviolet/visible spectrum are similar to LAM, and the enzyme requires SAM and dithionite as activators, as does LAM. Freeze-quench EPR experiments in the presence of l-glutamate reveal a glutamate-based free radical in the steady state of the reaction. A number of other bacterial genomes include genes encoding proteins homologous to the glutamate 2,3-aminomutase from C. difficile, and four of these proteins display the activity of glutamate 2,3-aminomutase when produced in E. coli. All of the homologous proteins have the cysteine motif CSMYCRHC corresponding to the motif CxxxCxxC characteristic of radical SAM enzymes. It is concluded that glutamate 2,3-aminomutase from C. difficile is a representative of a family found in a number of bacteria. It is likely that the beta-glutamate found in a few bacterial and archeal species as an osmolyte arises from the action of glutamate 2,3-aminomutase.

Basis for the equilibrium constant in the interconversion of l-lysine and l-beta-lysine by lysine 2,3-aminomutase.

l-beta-lysine and beta-glutamate are produced by the actions of lysine 2,3-aminomutase and glutamate 2,3-aminomutase, respectively. The pK(a) values have been titrimetrically measured and are for l-beta-lysine: pK(1)=3.25 (carboxyl), pK(2)=9.30 (beta-aminium), and pK(3)=10.5 (epsilon-aminium). For beta-glutamate the values are pK(1)=3.13 (carboxyl), pK(2)=3.73 (carboxyl), and pK(3)=10.1 (beta-aminium). The equilibrium constants for reactions of 2,3-aminomutases favor the beta-isomers. The pH and temperature dependencies of K(eq) have been measured for the reaction of lysine 2,3-aminomutase to determine the basis for preferential formation of beta-lysine. The value of K(eq) (8.5 at 37 degrees C) is independent of pH between pH 6 and pH 11; ruling out differences in pK-values as the basis for the equilibrium constant. The K(eq)-value is temperature-dependent and ranges from 10.9 at 4 degrees C to 6.8 at 65 degrees C. The linear van't Hoff plot shows the reaction to be enthalpy-driven, with DeltaH degrees =-1.4 kcal mol(-1) and DeltaS degrees =-0.25 cal deg(-1) mol(-1). Exothermicity is attributed to the greater strength of the bond C(beta)-N(beta) in l-beta-lysine than C(alpha)-N(alpha) in l-lysine, and this should hold for other amino acids.

Cloning, expression, purification, cofactor requirements, and steady state kinetics of phosphoketolase-2 from Lactobacillus plantarum.

The genes xpk1 and xpk2(Delta1-21) encoding phosphoketolase-1 and (Delta1-7)-truncated phosphoketolase-2 have been cloned from Lactobacillus plantarum and expressed in Escherichia coli. Both gene-products display phosphoketolase activity on fructose-6-phosphate in extracts. A N-terminal His-tag construct of xpk2(Delta1-21) was also expressed in E. coli and produced active His-tagged (Delta1-7)-truncated phosphoketolase-2 (hereafter phosphoketolase-2). Phosphoketolase-2 is activated by thiamine pyrophosphate (TPP) and the divalent metal ions Mg(2+), Mn(2+), or Ca(2+). Kinetic analysis and data from the literature indicate the activators are MgTPP, MnTPP, or CaTPP, and these species activate by an ordered equilibrium binding pathway, with Me(2+)TPP binding first and then fructose-6-phosphate. Phosphoketolase-2 accepts either fructose-6-phosphate or xylulose-5-phosphate as substrates, together with inorganic phosphate, to produce acetyl phosphate and either erythrose-4-phosphate or glyceraldehyde-3-phosphate, respectively. Steady state kinetic analysis of acetyl phosphate formation with either substrate indicates a ping pong kinetic mechanism. Product inhibition patterns with erythrose-4-phosphate indicate that an intermediate in the ping pong mechanism is formed irreversibly. Background mechanistic information indicates that this intermediate is 2-acetyl-TPP. The irreversibility of 2-acetyl-TPP formation might explain the overall irreversibility of the reaction of phosphoketolase-2.

Probing interactions from solvent-exchangeable protons and monovalent cations with the 1,2-propanediol-1-yl radical intermediate in the reaction of dioldehydrase.

The reaction of adenosylcobalamin-dependent dioldehydrase with 1,2-propanediol gives rise to a radical intermediate observable by EPR spectroscopy. This reaction requires a monovalent cation such as potassium ion. The radical signal arises from the formation of a radical pair comprised of the Co(II) of cob(II)alamin and a substrate-related radical generated upon hydrogen abstraction by the 5'-deoxyadenosyl radical. The high-field asymmetric doublet arising from the organic radical has allowed investigation of its composition and environment through the use of EPR spectroscopic techniques. To characterize the protonation state of the oxygen substituents in the radical intermediate, X-band EPR spectroscopy was performed in the presence of D(2)O and compared to the spectrum in H(2)O. Results indicate that the unpaired electron of the steady-state radical couples to a proton on the C(1) hydroxyl group. Other spectroscopic experiments were performed, using either potassium or thallous ion as the activating monovalent cation, in an attempt to exploit the magnetic nature of the (205,203)Tl nucleus to identify any intimate interaction of the radical intermediate with the activating cation. The radical intermediate in complex with dioldehydrase, cob(II)alamin and one of the activating monovalent cations was observed using EPR, ENDOR, and ESEEM spectroscopy. The spectroscopic evidence did not implicate a direct coordination of the activating cation and the substrate derived radical intermediate.

Serine Protease Catalysis: A Computational Study of Tetrahedral Intermediates and Inhibitory Adducts.

Peptide boronic acids and peptidyl trifluoromethyl ketones (TFKs) inhibit serine proteases by forming monoanionic, tetrahedral adducts to serine in the active sites. Investigators regard these adducts as analogs of monoanionic, tetrahedral intermediates. Density functional theory (DFT) calculations and fractional charge analysis show that tetrahedral adducts of model peptidyl TFKs are structurally and electrostatically very similar to corresponding tetrahedral intermediates. In contrast, the DFT calculations show the structures and electrostatic properties of analogous peptide boronate adducts to be significantly different. The peptide boronates display highly electrostatically positive boron, with correspondingly negative ligands in the tetrahedra. In addition, the computed boron-oxygen and boron-carbon bond lengths in peptide boronates (which are identical or very similar to the corresponding bonds in a peptide boronate adduct of α-lytic protease determined by X-ray crystallography at subangstrom resolution) are significantly longer than the corresponding bond lengths in model tetrahedral intermediates. Since protease-peptidyl TFKs incorporate low-barrier hydrogen bonds (LBHBs) between an active site histidine and aspartate, while the protease-peptide boronates do not, these data complement the spectroscopic and chemical evidence for the participation of LBHBs in catalysis by serine proteases. Moreover, while the potency of these classes of inhibitors can be correlated to the structures of the peptide moieties, the present results indicate that the strength of their bonds to serine contribute significantly to their inhibitory properties.

Correlation of Electronic Effects in N-Alkylnicotinamides with NMR Chemical Shifts and Hydride Transfer Reactivity.

The (13)C and (15)N NMR chemical shifts for ring atoms of a series of N-alkylnicotinamides are shown to be correlated with their reduction potentials and reactivities toward NaBH(3)CN. The nicotinamide compounds include N-ethyl-N-benzyl-N-[p-(trifluoromethyl)benzyl]-, N-(p-cyanobenzyl)-, N-(carbomethoxymethyl)-, and N-(cyanomethyl)nicotinamides. The values of delta()13(C) for all the ring carbons increase with increasing electron-withdrawing power of the N-alkyl substituent. The value for C-4 increases the most, a range of 2.4 ppm in this series, whereas those for other atoms increase on the order of 1 ppm. The value of delta()15(N) for N-1 decreases with increasing electron-withdrawing power over a range of 20 ppm. The NMR data indicate that inductive electron withdrawal by N-alkyl substituents polarizes the pi-electron system to decrease electron density on ring carbons and increase electron density on the ring nitrogen. The values of log k (second order) for reduction of these compounds by NaBH(3)CN are proportional to the values of delta()13(C) for C-4 and inversely proportional to delta()15(N) for N-1. The reduction potentials are proportional to delta()13(C). The substituent effects are qualitatively similar to the substrate-induced electrostatic effects on the nicotinamide ring of NAD(+) at the active site of UDP-galactose 4-epimerase (Burke, J. R.; Frey, P. A. Biochemistry 1993, 32, 13220-13230). However, they differ quantitatively in that the upfield perturbation at N-1 is smaller in the enzyme and the signal for C-6 is also shifted upfield. The substrate-induced enzymatic perturbation of electron density at C-4 of NAD(+) quantitatively accounts for its increase in reactivity at the active site, but the perturbation at N-1 is less closely correlated with reactivity.

Advances in research on metalloproteins.

Metal ions play essential roles in biological processes. Ions such as K(+) and Na(+) are important in ion transport, and Mg(2+), Ca(2+), and Zn(2+) are important chelators in many processes, including phosphotransfer and harvesting of light for energy metabolism. The transition metals readily undergo one-electron chemistry, and in this capacity they function uniquely in biological processes such as long-range and inner sphere electron transfer. They also facilitate many one-electron chemical reactions involving free radical intermediates. Iron, being the dominant element in the earth and a transition metal, most frequently participates in biological one-electron chemistry.

The ubiquity of iron.

The importance of iron in living systems can be traced to the many complexes within which it is found, to its chemical mobility in undergoing oxidation-reduction reactions, and to the abundance of iron in Earth's crust. Iron is the most abundant element, by mass, in the Earth, constituting about 80% of the inner and outer cores of Earth. The molten outer core is about 8000 km in diameter, and the solid inner core is about 2400 km in diameter. Iron is the fourth most abundant element in Earth's crust. It is the chemically functional component of mononuclear iron complexes, dinuclear iron complexes, [2Fe-2S] and [4Fe-4S] clusters, [Fe-Ni-S] clusters, iron protophorphyrin IX, and many other complexes in protein biochemistry. Metals such as nickel, cobalt, copper, and manganese are present in the crust and could in principle function chemically in place of iron, but they are scarce in Earth's crust. Iron is plentiful because of its nuclear stability in stellar nuclear fusion reactions. It seems likely that other solid planets, formed by the same processes as Earth, would also foster the evolution of life and that iron would be similarly important to life on those planets as it is on Earth.

Kinetic and spectroscopic evidence of negative cooperativity in the action of lysine 2,3-aminomutase.

Lysine 2,3-aminomutase (LAM) catalyzes the interconversion of L-lysine and L-ß-lysine, a component of a number of antibiotics. The reaction requires the cofactors S-adenosyl-L-methionine (SAM), pyridoxal-5'-phosphate (PLP), and a [4Fe-4S] cluster. LAM is a founding member of the radical SAM superfamily of enzymes. LAM is highly specific for L-lysine and will not accept most other amino acids as substrates. L-alanine and L-2-aminobutyrate at 0.2 M react as substrates for LAM at, respectively, 5 × 10(-6) and 8 × 10(-5) times the rate with saturating L-lysine. Saturating ethylamine accelerates the L-alanine reaction 70-fold, and saturating methylamine accelerates the L-2-aminobutyrate reaction 47-fold. The primary amines binding at the active site of LAM with L-alanine or L-2-aminobutyrate simulate L-lysine. The steady-state kinetics of the reaction of L-alanine + ethylamine displays negative cooperativity with respect to L-alanine. The second-order rate constant for production of ß-alanine in the reaction of L-alanine and saturating ethylamine is 0.040 M(-1) s(-1), which is 2 × 10(-5) times the value of k(cat)/K(m) for the reaction of L-lysine. When L-lysine is at a concentration 1/16th of K(m), the lysyl-free radical intermediate is hardly detectable by EPR; however, the addition of L-alanine at high concentration (0.2 M) enhances free radical formation, and the addition of ethylamine further enhances radical formation. These facts complement the kinetic observations and support negative cooperativity in the reaction of L-alanine as a substrate for LAM. Present results and independent evidence support negative cooperativity in the reaction of L-lysine as well.