Systematics of 2H patterns in natural compounds and its importance for the elucidation of biosynthetic pathways

The relative global ²H-content of natural plant products is correlated to that of the primary hydrogen source, i.e. water, to the site of their biosynthesis (C₃-, C₄- and CAM-plants; chloroplasts, cytosol), and to their biosynthetic pathways. A relative global ²H-content sequence can be established in the order phenylpropanoids > carbohydrates > bulk material > hydrolysable lipids > steroids. A detailed analysis of the ²H-patterns of the main groups of secondary compounds reveals regularities, in that they are correlated to the primary precursors and to the origin of hydrogen from four main pools with the mean δ²H-values [‰]_V?SMOW: leaf H₂O ～+30; carbohydrates ～?70; NADPH ～?250; flavoproteins ～?350. Aside from the ²H-discrimination between these pools, kinetic isotope effects on defined reactions only become effective in connection with metabolic branching events. So, the ²H-pattern of natural aromatic compounds can be correlated to the ²H-pattern of the precursor carbohydrates and a reduction step in the course of the shikimic acid pathway, furthermore to the implication of the NIH-shift. The pattern of aromatic compounds from the polyketide is different from that of the shikimate pathway. The alternating ²H-abundance of fatty acid chains is caused by the origin of their hydrogen atoms from carbohydrates and from NADPH, directly or via a flavoprotein, respectively. This is similar for isoprenoids, and the natural ²H-patterns permit their assignment to the mevalonate or non-mevalonate biosynthetic pathway. Generally, the correlations and regularities of the ²H-patterns of organic compounds found are a new reliable tool for the elucidation of biosynthetic pathways and origin assignments.     

Application of 2H NMR to the study of natural site-specific hydrogen isotope transfer among substrate,medium, and glycerol in glucose fermentation with yeast

2H NMR is a very useful tool in isotope tracing studies. This technique was applied to a quantitative study of a site-specific deuterium affiliation among the substrate, the medium, and a product (glycerol), in glucose fermentation with yeast. The quality of the results depends on the quantitative 2H NMR analysis of glycerol. After comparing several potential analysis probe molecules, the derivative of glycerol, 2,2-dimethyl-1,3-dioxolane-4-methanol, was chosen as the most advantageous. Using this probe in a set of isotope-labeling experiments, we describe how a complete quantitative site-specific hydrogen isotope transfer model, which connects the site-specific isotopic ratios of the substrate, the medium, and the products, can be established. This model can provide information on complex hydrogen transfer mechanisms during biochemical reactions and can be useful for the prediction of site-specific hydrogen isotopic ratios at natural abundance of the products, based on that of the substrate or reactants and the medium.     

Automated extraction of backbone deuteration levels from amide H/2H mass spectrometry experiments

A Fourier deconvolution method has been developed to explicitly determine the amount of backbone amide deuterium incorporated into protein regions or segments by hydrogen/deuterium (H/D) exchange with high-resolution mass spectrometry. Determination and analysis of the level and number of backbone amide exchanging in solution provide more information about the solvent accessibility of the protein than do previous centroid methods, which only calculate the average deuterons exchanged. After exchange, a protein is digested into peptides as a way of determining the exchange within a local area of the protein. The mass of a peptide upon deuteration is a sum of the natural isotope abundance, fast exchanging side-chain hydrogens (present in MALDI-TOF H/2H data) and backbone amide exchange. Removal of the components of the isotopic distribution due to the natural isotope abundances and the fast exchanging side-chains allows for a precise quantification of the levels of backbone amide exchange, as is shown by an example from protein kinase A. The deconvoluted results are affected by overlapping peptides or inconsistent mass envelopes, and evaluation procedures for these cases are discussed. Finally, a method for determining the back exchange corrected populations is presented, and its effect on the data is discussed under various circumstances.     

Peroxidase-catalyzed N-demethylation reactions. Substrate deuterium isotope effects

Deuterium isotope effects on the kinetic parameters for the hydroperoxide-supported N-demethylation of N,N-dimethylaniline catalyzed by chloroperoxidase and horseradish peroxidase were determined using N,N-di-(trideuteromethyl)aniline. The isotope effect on the Vmax for the chloroperoxidase-catalyzed demethylation reaction supported by ethyl hydroperoxide was 1.42 +/- 0.31. The isotope effects on the Vmax for the horseradish peroxidase-catalyzed reaction supported by ethyl hydroperoxide and hydrogen peroxide were 1.99 +/- 0.39 and 4.09 +/- 0.27, respectively. Isotope effects ranging from 1.76 to 5.10 were observed on the Vmax/Km for the hydroperoxide substrate (i.e. the second order rate constant for the reaction of the hydroperoxide with the peroxidase to form compound I) in both enzyme systems when the N-methyl groups of N,N-dimethylaniline were deuterated. These results are not predicted by the simple ping-pong kinetic model for peroxidase-catalyzed N-demethylation reactions. The data are most simply explained by a mechanism involving the transfer of deuterium (or hydrogen) from N,N-dimethylaniline to the enzyme during catalysis. The deuterium must subsequently be displaced from the enzyme by the hydroperoxide, causing the observed isotope effects.     

Isolation of a Bacillus sp. capable of transforming isoeugenol to vanillin

Natural aroma compounds are of major interest to the flavor and fragrance industry. Due to the limited sources for natural aromas, there is a growing interest in developing alternative sources for natural aroma compounds, and in particular aromatic aldehydes. In several microbial species aromatic aldehydes are detected as intermediates in the degradation pathway of phenylpropanoids. Thus, bioconversion of phenylpropanoids is one possible route for the production of these aroma compounds. The present work describes the isolation of microbial strains, capable of producing vanillin from isoeugenol. Bacterial strains isolated from soil, were screened for their ability to transform isoeugenol to vanillin. One of these strains, strain B2, was found to produce high amounts of vanillin when grown in the presence of isoeugenol, and was also capable of growing on isoeugenol as the sole carbon source. Based on its fatty acids profile, strain B2 was identified as a Bacillus subtilis sp. The bioconversion capabilities of strain B2 were tested in growing cultures and cell free extracts. In the presence of isoeugenol, a growing cultures of B. subtilis B2 produced 0.61 g l-1 vanillin (molar yield of 12.4%), whereas cell free extracts resulted in 0.9 g l-1 vanillin (molar yield of 14%).     

Kinetic deuterium isotope effects on the N-demethylation of tertiary amides by cytochrome P-450

Liver microsomal cytochrome P-450 readily N-dealkylates N,N-dimethylamides. N-Methyl-N-hydroxymethyl amides were isolated as intermediates and characterized by gas chromatography-mass spectrometry as their trimethylsilyl ethers. Intramolecular kinetic deuterium isotope effects measured for the enzymic N-demethylation of a series of 12 aromatic and aliphatic N-methyl-N-trideuteriomethyl amides, RCON(CH3)CD3, varied from 3.6 to 6.9 but were independent of both amide bond rotation rate and substrate oxidation potential. These values, which represent a lower limit to the intrinsic isotope effect (Dkintrinsic), are significantly larger than those observed for anodic N-demethylation and are consistent with a mechanism involving hydrogen atom abstraction. On the other hand, with N,N-dimethylbenzamide the intermolecular kinetic deuterium isotope effects on Vmax and Vmax/Km were found to be much smaller (1.23 and 1.75, respectively) indicating substantial suppression of the intrinsic isotope effect. Such suppression indicates the occurrence of a rate-limiting step other than the isotopically sensitive step together with a strong commitment to catalysis.     

Quantitative isotopic C nuclear magnetic resonance at natural abundance to probe enzyme reaction mechanisms via site-specific isotope fractionation: The case of the chain-shortening reaction for the bioconversion of ferulic acid to vanillin

Isotope fractionation is a powerful technique by which to probe the reaction mechanism of enzymes. The effect of a heavy isotope on the reaction energetics can be used to predict transition state architecture and reaction mechanism. In order to examine simultaneously the isotope fractionation in ¹³C at multiple sites within the substrate and product molecules without any need for site-selective isotope enrichment, a technique exploiting quantitative isotopic nuclear magnetic resonance (NMR) spectrometry at natural abundance (NAQ–NMR) has been developed. Here we report the first application of this technique to the study of an enzyme-catalyzed reaction, the bioconversion of ferulic acid to vanillin in cultures of Streptomyces setonii. We were able to show that the NAQ–NMR methodology is sufficiently precise and robust to measure the isotope shifts in the ¹³C/¹²C ratios in both substrate and product of this biotransformation, thereby permitting meaningful data to be obtained even at carbon positions that take part only indirectly in the reaction and show only secondary isotope fractionation. The results obtained provide direct evidence in support of the current hypothesis for the reaction mechanism of the enzyme hydroxycinnamoyl–CoA hydratase/lyase, notably the proposed involvement of the quinone methide enolate of feruloyl–CoA as intermediate in the catalytic pathway.     

Stereochemistry of the enolization of scytalone by scytalone dehydratase

In D(2)O, scytalone exchanges its two C2 hydrogen atoms for deuterium atoms at different rates. At pD 7.0 and 25 degrees C, half-lives for the exchanges are 0.8 and 10 days for the pro-S and pro-R hydrogens, respectively. The differential exchange rates allow for the preparation of multiple scytalone samples (through incubation of scytalone in D(2)O and then back exchanging with H(2)O) having differential levels of deuterium enrichment at the C2 pro-S and pro-R positions. From these samples, the stereochemical preference for hydrogen abstraction during the dehydration reaction mediated by the enzyme scytalone dehydratase was determined. At pH 7. 0, deuterium at the pro-S position has little effect on enzyme catalysis, whereas deuterium at the pro-R position produces kinetic isotope effects of 2.3 (25 degrees C), 5.1 (25 degrees C), and 6.7 (6.8 degrees C) on k(cat), k(cat)/K(m), and the single-turnover rate, respectively. The results are fully consistent with the enzyme catalyzing a syn elimination through an E1cb-like mechanism. The syn elimination is compatible with the interactions realized between a scytalone boat conformation and key active site residues as modeled from multiple X-ray crystal structures of the enzyme in complexes with inhibitors.     

Kinetic isotope effects on aromatic and benzylic hydroxylation by Chromobacterium violaceum phenylalanine hydroxylase as probes of chemical mechanism and reactivity

Phenylalanine hydroxylase from Chromobacterium violaceum (CvPheH) is a non-heme iron monooxygenase that catalyzes the hydroxylation of phenylalanine to tyrosine. In this study, we used deuterium kinetic isotope effects to probe the chemical mechanisms of aromatic and benzylic hydroxylation to compare the reactivities of bacterial and eukaryotic aromatic amino acid hydroxylases. The (D) k cat value for the reaction of CvPheH with [(2)H 5]phenylalanine is 1.2 with 6-methyltetrahydropterin and 1.4 with 6,7-dimethyltetrahydropterin. With the mutant enzyme I234D, the (D) k cat value decreases to 0.9 with the latter pterin; this is likely to be the intrinsic effect for addition of oxygen to the amino acid. The isotope effect on the subsequent tautomerization of a dienone intermediate was determined to be 5.1 by measuring the retention of deuterium in tyrosine produced from partially deuterated phenylalanine; this large isotope effect is responsible for the normal effect on k cat. The isotope effect for hydroxylation of the methyl group of 4-CH 3-phenylalanine, obtained from the partitioning of benzylic and aromatic hydroxylation products, is 10. The temperature dependence of this isotope effect establishes the contribution of hydrogen tunneling to benzylic hydroxylation by this enzyme. The results presented here provide evidence that the reactivities of the prokaryotic and eukaryotic hydroxylases are similar and further define the reactivity of the iron center for the family of aromatic amino acid hydroxylases.     

Human erythrocyte glutathione reductase: chemical mechanism and structure of the transition state for hydride transfer

Kinetic parameters and primary deuterium kinetic isotope effects for NADH and five pyridine nucleotide substrates have been determined at pH 8.1 for human erythrocyte glutathione reductase. DV/KNADH and DV are equal to 1.4 and are pH independent below pH 8.1, but DV decreases to 1.0 at high pH as a group exhibiting a pK of 8.6 is deprotonated. This result suggests that as His-467' is deprotonated, the rate of the isotopically insensitive oxidative half-reaction is specifically decreased and becomes rate-limiting. For all substrates, equivalent V and V/K primary deuterium kinetic isotope effects are observed at pH values below 8.1. The primary deuterium kinetic isotope effect on V, but not V/K, is sensitive to solvent isotopic composition. The primary tritium kinetic isotope effects agree well with the corresponding value calculated from the primary deuterium kinetic isotope effects by using the Swain-Schaad relationship. This suggests that the primary deuterium kinetic isotope effects observed in these steady-state experiments are the intrinsic primary deuterium kinetic isotope effects for hydride transfer. The magnitude of the primary deuterium kinetic isotope effect is dependent on the redox potential of the pyridine nucleotide substrate used, varying from approximately 1.4 for NADH and -320 mV reductants to 2.7 for thioNADH to 4.2-4.8 for 3-acetylpyridine adenine dinucleotide (3APADH). The alpha-secondary tritium kinetic isotope effects also increase as the redox potential of the pyridine nucleotide substrate becomes more positive. Together, these data indicate that the transition state for hydride transfer is very early for NADH and becomes later for thioNADH and 3APADH, as predicted by Hammond's postulate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of the reaction by which natural vitamin E is regenerated by vitamin C

The rate constant and activation energy of the regeneration reaction of natural vitamin E by vitamin C were determined with a double-mixing stopped-flow spectrophotometer. The formation of vitamin C radical was observed in the absorption spectrum. The kinetic effect of methyl substitution on the aromatic ring of vitamin E radical indicates that partial charge-transfer plays a role in the reaction. Since a substantial deuterium kinetic isotope effect was not found, the tunneling effect may not play an important role under the present experimental conditions.     

Site-specific isotope fractionation of hydrogen in the biosynthesis of plant fatty acids

The overall deuterium content of plant lipids has been investigated by isotope ratio mass spectrometry (IRMS), and the site-specific natural isotope fractionation of hydrogen has been studied by ²H-NMR at natural abundance (SNIF-NMR). An analytical strategy has been developed in order to exploit the isotopomeric composition determined in clusters associated with different chemical sites of one or several fatty acid components. The method, which combines spectrometric and chromatographic data, enables isotopic criteria to be directly derived from raw vegetable oils containing in general two saturated and two unsaturated fatty acids. These results provide new information on isotopic fractionation caused by biochemical, physiological and natural environmental effects. Some alternation in the molecular deuterium distribution has been detected which may be related to the mechanism of fatty acid elongation. The successive methylene groups introduced through malonyl CoA are the subjects of different kinetic isotope effects since one of them is exclusively derived from NADH whereas the other has a contribution from pyruvate. A discriminant analysis of the cluster isotopic parameters enables several kinds of botanical precursors to be distinguished. The authenticating performances can be improved by taking into account the influence of climatic effects related to the region in which the plant grew.     

Hydrogen peroxide elimination from C4a-hydroperoxyflavin in a flavoprotein oxidase occurs through a single proton transfer from flavin N5 to a peroxide leaving group

C4a-hydroperoxyflavin is found commonly in the reactions of flavin-dependent monooxygenases, in which it plays a key role as an intermediate that incorporates an oxygen atom into substrates. Only recently has evidence for its involvement in the reactions of flavoprotein oxidases been reported. Previous studies of pyranose 2-oxidase (P2O), an enzyme catalyzing the oxidation of pyranoses using oxygen as an electron acceptor to generate oxidized sugars and hydrogen peroxide (H(2)O(2)), have shown that C4a-hydroperoxyflavin forms in P2O reactions before it eliminates H(2)O(2) as a product (Sucharitakul, J., Prongjit, M., Haltrich, D., and Chaiyen, P. (2008) Biochemistry 47, 8485-8490). In this report, the solvent kinetic isotope effects (SKIE) on the reaction of reduced P2O with oxygen were investigated using transient kinetics. Our results showed that D(2)O has a negligible effect on the formation of C4a-hydroperoxyflavin. The ensuing step of H(2)O(2) elimination from C4a-hydroperoxyflavin was shown to be modulated by an SKIE of 2.8 ± 0.2, and a proton inventory analysis of this step indicates a linear plot. These data suggest that a single-proton transfer process causes SKIE at the H(2)O(2) elimination step. Double and single mixing stopped-flow experiments performed in H(2)O buffer revealed that reduced flavin specifically labeled with deuterium at the flavin N5 position generated kinetic isotope effects similar to those found with experiments performed with the enzyme pre-equilibrated in D(2)O buffer. This suggests that the proton at the flavin N5 position is responsible for the SKIE and is the proton-in-flight that is transferred during the transition state. The mechanism of H(2)O(2) elimination from C4a-hydroperoxyflavin is consistent with a single proton transfer from the flavin N5 to the peroxide leaving group, possibly via the formation of an intramolecular hydrogen bridge.     

Experimental Evidence for a Hydride Transfer Mechanism in Plant Glycolate Oxidase Catalysis

In plants, glycolate oxidase is involved in the photorespiratory cycle, one of the major fluxes at the global scale. To clarify both the nature of the mechanism and possible differences in glycolate oxidase enzyme chemistry from C₃ and C₄ plant species, we analyzed kinetic parameters of purified recombinant C₃ (Arabidopsis thaliana) and C₄ (Zea mays) plant enzymes and compared isotope effects using natural and deuterated glycolate in either natural or deuterated solvent. The ¹²C/¹³C isotope effect was also investigated for each plant glycolate oxidase protein by measuring the ¹³C natural abundance in glycolate using natural or deuterated glycolate as a substrate. Our results suggest that several elemental steps were associated with an hydrogen/deuterium isotope effect and that glycolate α-deprotonation itself was only partially rate-limiting. Calculations of commitment factors from observed kinetic isotope effect values support a hydride transfer mechanism. No significant differences were seen between C₃ and C₄ enzymes.     

Transformation of ortho-substituted biphenyls by Methylosinus trichosporium OB3b: substituent effects on oxidation kinetics and product formation

The ability of Methylosinus trichosporium OB3b, expressing soluble methane monooxygenase, to oxidize a range of ortho-substituted biphenyls was examined to better understand how substituents affect both the rate and products of oxidation in comparison to biphenyl. Inhibition of oxidation was observed over the tested substrate range for both biphenyl and ortho-halogenated biphenyls (2-chloro-, 2-bromo-, and 2-iodobiphenyl). No inhibition was observed during the oxidation of 2-hydroxybiphenyl and 2-methylbiphenyl. Analysis of the products of oxidation showed that, depending on the substituent, ring hydroxylation, substituent oxidation, and elimination pathways could occur. The type and abundance of products formed along with the relatively high kinetic isotope effect observed for deuterated vs. nondeuterated biphenyl (k(h)/k(d) = 3.4+/-0.02) are consistent with mechanisms that include both hydrogen abstraction and NIH-shift pathways. Knowledge of these substituent-dependent reaction rates and mechanisms enhances our understanding of the methanotrophic aryl transformation potential and allows for better prediction of the formation of oxidized intermediates by methanotrophic bacteria.     

Mechanistic characterization of omega-3 desaturation in the green alga Chlorella vulgaris

alpha-Linolenic acid (ALA, 9(Z),12(Z),15(Z)-octadecatrienoic acid) derivatives are important plant lipids which play a critical key role in cold tolerance. The final steps of ALA biosynthesis feature a series of regio- and stereoselective dehydrogenation reactions which are catalyzed by a set of enzymes known as fatty acid desaturases. In conjunction with ongoing research into the structural biology of these remarkable catalysts, we have examined the mechanism of double bond introduction at C15,16 as it occurs in a model photosynthetic organism, Chlorella vulgaris. The individual deuterium kinetic isotope effects associated with the C-H bond cleavages at C-15 and C-16 of a thialinoleoyl analogue were measured via competition experiments using appropriately deuterium-labelled 7-thia substrates. A large kinetic isotope effect (KIE) (k(H)/k(D)=10.2+/-2.8) was observed for the C-H bond-breaking step at C-15 while the C-H bond cleavage at C-16 was found to be relatively insensitive to deuterium substitution (k(H)/k(D)=0.8+/-0.2). These results point to C-15 as the site of initial oxidation in omega-3 desaturation and imply that the Chlorella and corresponding plant systems share a common active site architecture.     

Three types of stereospecificity and the kinetic deuterium isotope effect in the oxidative deamination of dopamine as catalyzed by different amine oxidases

When the stereospecifically deuterated dopamine enantiomers, (R)- and (S)-[alpha-2H1]dopamine, are incubated with amine oxidases, the deuterium atom may be either retained to form monodeuterated 3,4-dihydroxyphenylacetaldehyde, or eliminated to produce the nondeuterated or protio-aldehyde product. These two aldehydes can be separated from one another and identified by high-performance liquid chromatography with electrochemical detection. Three types of stereospecific abstraction of a hydrogen from the alpha-carbon of dopamine during deamination have been observed. In the first type, the pro-R hydrogen is removed from the alpha-carbon. Enzymes in this category are mitochondrial monoamine oxidases A and B, as isolated from different tissues and species. The second type of deamination involves the abstraction of pro-S hydrogen from the alpha-carbon of dopamine. Soluble enzymes, such as rat aorta benzylamine oxidase or diamine oxidase from hog kidney and pea seedling, have been found to belong to this group. Bovine plasma amine oxidase exhibits the third type of deamination where no absolute stereospecificity is required. This enzyme catalyzes the oxidation of either (S)- or (R)-[alpha-2H1]dopamine, preferably breaking the C-H bond rather than the C-2H bond in both cases. The kinetic deuterium isotope effect during the deamination of dopamine catalyzed by the different amine oxidases varies greatly; VH/VD ranges from 1.5 to 5.5. The high magnitude of the isotope effect suggests that hydrogen abstraction may be the rate-limiting step (i.e., in reactions catalyzed by benzylamine oxidase and monoamine oxidase). When the isotope effect is low (i.e., for diamine oxidases from hog kidney or pea seedling), it is uncertain if the breaking of the bond is rate limiting.     

Small amounts of isotope-reinforced polyunsaturated fatty acids suppress lipid autoxidation

Polyunsaturated fatty acids (PUFAs) undergo autoxidation and generate reactive carbonyl compounds that are toxic to cells and associated with apoptotic cell death, age-related neurodegenerative diseases, and atherosclerosis. PUFA autoxidation is initiated by the abstraction of bis-allylic hydrogen atoms. Replacement of the bis-allylic hydrogen atoms with deuterium atoms (termed site-specific isotope-reinforcement) arrests PUFA autoxidation due to the isotope effect. Kinetic competition experiments show that the kinetic isotope effect for the propagation rate constant of Lin autoxidation compared to that of 11,11-D(2)-Lin is 12.8 ± 0.6. We investigate the effects of different isotope-reinforced PUFAs and natural PUFAs on the viability of coenzyme Q-deficient Saccharomyces cerevisiae coq mutants and wild-type yeast subjected to copper stress. Cells treated with a C11-BODIPY fluorescent probe to monitor lipid oxidation products show that lipid peroxidation precedes the loss of viability due to H-PUFA toxicity. We show that replacement of just one bis-allylic hydrogen atom with deuterium is sufficient to arrest lipid autoxidation. In contrast, PUFAs reinforced with two deuterium atoms at mono-allylic sites remain susceptible to autoxidation. Surprisingly, yeast treated with a mixture of approximately 20%:80% isotope-reinforced D-PUFA:natural H-PUFA are protected from lipid autoxidation-mediated cell killing. The findings reported here show that inclusion of only a small fraction of PUFAs deuterated at the bis-allylic sites is sufficient to profoundly inhibit the chain reaction of nondeuterated PUFAs in yeast.     

Deuterium isotope effects in carbohydrates revisited. Cryoprobe studies of the anomerization and NH to ND deuterium isotope induced 13C NMR chemical shifts of acetamidodeoxy and aminodeoxy sugars

Complete ¹H and ¹³C NMR chemical shift assignments have been generated from a series of acetamidodeoxy and aminodeoxy sugar derivatives. For free sugars, the enhanced sensitivity of an NMR cryoprobe allowed simple 1D and 2D NMR spectra to be obtained from essentially single anomers, before significant mutarotation had occurred. The NMR assignments have been used to characterize deuterium isotope effects on ¹³C chemical shifts measured under conditions of slow NH to ND exchange in single solutions. Within a range of 0 to −0.138 ppm, β, γ, δ, and ζ deuterium isotope effects have been observed, thus providing additional reference data for assignment of the ¹³C NMR spectra of nitrogenous saccharides.     

Kinetic and biochemical analysis of the mechanism of action of lysine 5,6-aminomutase

Lysine 5,6-aminomutase (5,6-LAM) catalyzes the reversible and nearly isoenergetic transformations of D-lysine into 2,5-diaminohexanoate (2,5-DAH) and of L-beta-lysine into 3,5-diaminohexanoate (3,5-DAH). The activity of 5,6-LAM depends on pyridoxal-5(')-phosphate (PLP) and adenosylcobalamin. The currently postulated multistep mechanism involves at least 12 steps, two of which involve hydrogen transfer. The deuterium kinetic isotope effects on k(cat) and k(cat)/K(m) have been found to be 10.4+/-0.3 and 8.3+/-1.9, respectively, in the reaction of DL-lysine-3,3,4,4,5,5,6,6-d(8). The corresponding isotope effects for reaction of DL-lysine-4,4,5,5-d(4) are 8.5+/-0.7 and 7.1+/-1.2, respectively. Neither cob(II)alamin nor a free radical can be detected in the steady state by UV-Vis spectrophotometry or electron paramagnetic resonance (EPR) spectroscopy. Therefore, hydrogen abstraction from carbon-5 of the substrate side chain is rate limiting in the mechanism. DL-4-Oxalysine is an alternative substrate for 5,6-LAM. DL-4-Oxalysine reacts irreversibly because the product breaks down into ammonia, acetaldehyde, and DL-serine. The value of K(m) for the reaction of DL-4-oxalysine is lower than that for DL-lysine and that of k(cat) for DL-4-oxalysine is slightly lower than that for DL-lysine. As measured by values of k(cat)/K(m), 5,6-LAM uses DL-4-oxalysine essentially as efficiently as the best substrates, D-lysine and L-beta-lysine, and more efficiently than DL-lysine. DL-4-Oxalysine induces the same suicide inactivation by electron transfer as do the biological substrates. The putative substrate-related radical intermediate is not sufficiently stabilized by the nonbonding 4-oxa electrons to be detectable by EPR spectroscopy.