Adenosylcobalamin-dependent Enzyme Research Articles

Steady-state and pre-steady state kinetic analysis of ornithine 4,5-aminomutase.

Ornithine 4,5-aminomutase (4,5-OAM) is a pyridoxal 5'-phosphate and adenosylcobalamin-dependent enzyme that catalyzes a 1,2-rearrangement of the terminal amine of d-ornithine to form (2R, 4S)-diaminopentanoate. The gene encoding ornithine 4,5-aminomutase is clustered with other genes that function in the oxidative l-ornithine metabolic pathway present in a number of anaerobic bacteria. This chapter discusses the methodology for measuring 4,5-OAM activity using NAD+-dependent diaminopentanoate dehydrogenase, which functions downstream of 4,5-OAM in the l-ornithine metabolic pathway. The use of ornithine racemace, which functions upstream of 4,5-OAM, for the synthesis of d,l-ornithine-3,3,4,4,5,5-d6 is also presented. Finally, this chapter describes the anaerobic stopped-flow spectrophotometric analysis of 4,5-OAM. Information is provided on the integration of a stopped-flow system in the anaerobically-maintained glove, the preparation of anaerobic solutions, and the experimental approach.

Using kinetic isotope effects to probe the mechanism of adenosylcobalamin-dependent enzymes.

Adenosylcobalamin- (AdoCbl) dependent enzyme reactions involved the transfer of hydrogen atoms between the 5'-carbon of the coenzyme and the substrates and products of the reaction. Tritium and deuterium kinetic isotope effect measurements are, therefore, a valuable tool to probe the mechanisms of AdoCbl-dependent enzymes, as they can provide information about the reaction pathway and the rate-determining step. Furthermore, if the intrinsic kinetic isotope effect can be isolated, information on the nature of the transition state associated with hydrogen transfer can be obtained. In this chapter I present methods for the preparation of isotopically-labeled AdoCbl and their use in rapid chemical quench experiments that allow isotope effects on specific steps in the reaction to be isolated. These techniques are illustrated with examples from my laboratory's studies on the AdoCbl dependent enzyme, glutamate mutase.

Metabolic Consequences of Cobalamin Scarcity in the Diatom Thalassiosira pseudonana as Revealed Through Metabolomics

Diatoms perform an estimated 20% of global photosynthesis, form the base of the marine food web, and sequester carbon into the deep ocean through the biological pump. In some areas of the ocean, diatom growth is limited by the micronutrient cobalamin (vitamin B12), yet the biochemical ramifications of cobalamin limitation are not well understood. In a laboratory setting, we grew the diatom Thalassiosira pseudonana under replete and low cobalamin conditions to elucidate changes in metabolite pools. Using metabolomics, we show that the diatom experienced a metabolic cascade under cobalamin limitation that affected the central methionine cycle, transsulfuration pathway, and composition of osmolyte pools. In T. pseudonana, 5'-methylthioadenosine decreased under low cobalamin conditions, suggesting a disruption in the diatom's polyamine biosynthesis. Furthermore, two acylcarnitines accumulated under low cobalamin, suggesting the limited use of an adenosylcobalamin-dependent enzyme, methylmalonyl CoA mutase. Overall, these changes in metabolite pools yield insight into the metabolic consequences of cobalamin limitation in diatoms and suggest that cobalamin availability may have consequences for microbial interactions that are based on metabolite production by phytoplankton.

A Novel Pathway for Bacterial Ethanolamine Metabolism

Ethanolamine, a major component of the cell membranes of bacteria and animals in the form of phosphatidylethanolamine, can be a source of carbon and/or nitrogen for many bacterial species. The canonical model for bacterial ethanolamine utilization (eut) requires the adenosylcobalamin-dependent enzyme ethanolamine ammonia-lyase, which catalyzes the conversion of ethanolamine to ammonia and acetaldehyde for cellular nitrogen and carbon, respectively. Here, we report discovery and characterization of a novel pathway for bacterial ethanolamine metabolism in organisms that lack canonical eut genes. We use molecular docking, enzymology, microbiology, and mass spectrometry to delineate a four-enzyme pathway for ethanolamine catabolism that terminates in glycine, a source of cellular nitrogen. Initially, a gamma-glutamyl synthetase catalyzes the ATP-dependent condensation of ethanolamine with L-glutamate to form γ-glutamyl-ethanolamide. Oxidation of the hydroxyl group of γ-glutamyl-ethanolamide by an alcohol dehydrogenase and subsequent oxidation of the resulting aldehyde by an aldehyde dehydrogenase leads to formation of γ-glutamyl glycine. Finally, hydrolysis of the γ-glutamyl glycine peptide bond by an amidohydrolase yields L-glutamate and glycine, which is assimilated as a nitrogen source. Using the Enzyme Function Initiative Enzyme Similarity Tool (EFI-EST) and Genome Neighborhood Tool (EFI-GNT), we show that a diverse set of organisms ranging from the Actinobacteria to the Proteobacteria possess the genetic capability for non-eut ethanolamine metabolism, with multiple apparent instances of non-orthologous gene displacement (NOD) between different phyla. This research was supported by US National Institutes of Health grant U54GM093342.

Non-canonical active site architecture of the radical SAM thiamin pyrimidine synthase.

Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 5′-deoxyadenosyl radical. Canonical radical SAM enzymes are characterized by a β-barrel-like fold and SAM anchors to the differentiated iron of the cluster, which is located near the amino terminus and within the β-barrel, through its amino and carboxylate groups. Here we show that ThiC, the thiamin pyrimidine synthase in plants and bacteria, contains a tethered cluster-binding domain at its carboxy terminus that moves in and out of the active site during catalysis. In contrast to canonical radical SAM enzymes, we predict that SAM anchors to an additional active site metal through its amino and carboxylate groups. Superimposition of the catalytic domains of ThiC and glutamate mutase shows that these two enzymes share similar active site architectures, thus providing strong evidence for an evolutionary link between the radical SAM and adenosylcobalamin-dependent enzyme superfamilies.

Entropic origin of cobalt-carbon bond cleavage catalysis in adenosylcobalamin-dependent ethanolamine ammonia-lyase.

Adenosylcobalamin-dependent enzymes accelerate the cleavage of the cobalt-carbon (Co-C) bond of the bound coenzyme by >10(10)-fold. The cleavage-generated 5'-deoxyadenosyl radical initiates the catalytic cycle by abstracting a hydrogen atom from substrate. Kinetic coupling of the Co-C bond cleavage and hydrogen-atom-transfer steps at ambient temperatures has interfered with past experimental attempts to directly address the factors that govern Co-C bond cleavage catalysis. Here, we use time-resolved, full-spectrum electron paramagnetic resonance spectroscopy, with temperature-step reaction initiation, starting from the enzyme-coenzyme-substrate ternary complex and (2)H-labeled substrate, to study radical pair generation in ethanolamine ammonia-lyase from Salmonella typhimurium at 234-248 K in a dimethylsulfoxide/water cryosolvent system. The monoexponential kinetics of formation of the (2)H- and (1)H-substituted substrate radicals are the same, indicating that Co-C bond cleavage rate-limits radical pair formation. Analysis of the kinetics by using a linear, three-state model allows extraction of the microscopic rate constant for Co-C bond cleavage. Eyring analysis reveals that the activation enthalpy for Co-C bond cleavage is 32 ± 1 kcal/mol, which is the same as for the cleavage reaction in solution. The origin of Co-C bond cleavage catalysis in the enzyme is, therefore, the large, favorable activation entropy of 61 ± 6 cal/(mol·K) (relative to 7 ± 1 cal/(mol·K) in solution). This represents a paradigm shift from traditional, enthalpy-based mechanisms that have been proposed for Co-C bond-breaking in B12 enzymes. The catalysis is proposed to arise from an increase in protein configurational entropy along the reaction coordinate.

Optimization and validation of a reversed-phase high performance liquid chromatography method for the measurement of bovine liver methylmalonyl-coenzyme a mutase activity

BackgroundMethylmalonyl-CoA mutase (MCM) is an adenosylcobalamin-dependent enzyme that catalyses the interconversion of (2R)-methylmalonyl-CoA to succinyl-CoA. In humans, a deficit in activity of MCM, due to an impairment of intracellular formation of adenosylcobalamin and methylcobalamin results in a wide spectrum of clinical manifestations ranging from moderate to fatal. Consequently, MCM is the subject of abundant literature. However, there is a lack of consensus on the reliable method to monitor its activity. This metabolic pathway is highly solicited in ruminants because it is essential for the utilization of propionate formed during ruminal fermentation. In lactating dairy cows, propionate is the major substrate for glucose formation. In present study, a reversed-phase high performance liquid chromatography (RP-HPLC) was optimized and validated to evaluate MCM activity in bovine liver. The major aim of the study was to describe the conditions to optimize reproducibility of the method and to determine stability of the enzyme and its product during storage and processing of samples.ResultsSpecificity of the method was good, as there was no interfering peak from liver extract at the retention times corresponding to methylmalonyl-CoA or succinyl-CoA. Repeatability of the method was improved as compared to previous RP-HPLC published data. Using 66 μg of protein, intra-assay coefficient of variation (CV) of specific activities, ranged from 0.90 to 8.05% and the CV inter-day was 7.40%. Storage and processing conditions (frozen homogenate of fresh tissue vs. fresh homogenate of tissue snapped in liquid nitrogen) did not alter the enzyme activity. The analyte was also stable in liver crude extract for three frozen/thawed cycles when stored at -20°C and thawed to room temperature.ConclusionsThe improved method provides a way for studying the effects of stages of lactation, diet composition, and physiology in cattle on MCM activity over long periods of time, such as a complete lactation period. Interestingly, this sensitive and accurate method could benefit the study of the cobalamin status in experimental studies and clinical cases.

Converging on a mechanism for choline degradation

In PNAS, Craciun and Balskus (1) combine biochemical intuition with modern genome mining techniques to discover the elusive enzyme responsible for catalyzing the degradation of choline ( 1 , Scheme 1). Choline is an essential nutrient in higher organisms that is required for the biosynthesis of the neurotransmitter acetylcholine and the head group of several phospholipids, and that serves as a source of the methyl groups for methionine and S -adenosylmethionine (2). In humans and other mammals, choline is catabolized to trimethylamine (TMA; 2 , Scheme 1) by symbiotic gut microbes, and irregularities in choline and TMA metabolism have been linked to liver and cardiovascular diseases, atherosclerosis, and deficiencies in fetal brain development (3⇓–5). TMA derived from choline is also a substrate for methanogenesis by marine microorganisms and, as such, contributes to global production of the greenhouse gas methane. Scheme 1. Mechanistic similarities in the catabolism of choline, ethanolamine, and glycerol; in each case, the bonds highlighted in red are cleaved by enzyme-mediated radical chemistry that results in a 1,2-migration. Scheme 2. Putative chemical mechanism of ethanolamine degradation by the adenosylcobalamin-dependent enzyme, ethanolamine ammonia lyase (EAL). To identify the unknown choline degrading enzyme, which has eluded researchers for more than a century, the authors postulated that the conversion of choline to TMA and acetaldehyde ( 3 , Scheme 1) may share mechanistic similarities to the well-studied catabolism of ethanolamine ( 4 , Scheme 1) to ammonia and acetaldehyde by bacterial enzymes encoded in the ethanolamine utilization ( eut ) gene cluster (6⇓–8). In ethanolamine degradation, C-N bond cleavage is carried out by the adenosylcobalamin (AdoCbl)-dependent enzyme, ethanolamine ammonia lyase (EAL), encoded by the eutBC genes (6). After the EAL-catalyzed cleavage of the C-N bond of … [↵][1]1To whom correspondence should be addressed. E-mail: vddonk{at}illinois.edu. [1]: #xref-corresp-1-1

Increasing complexity models for describing the generation of substrate radicals at the active site of ethanolamine ammonia-lyase/B 12

Ethanolamine ammonia-lyase ( EAL) is an adenosylcobalamin ( AdoCbl, a B 12 coenzyme) dependent enzyme expressed in enteric bacteria, which catalyzes conversion of ethanolamine in ammonia and acetaldehyde under anaerobic conditions. The most accepted reaction mechanism includes five steps: (a) 5-deoxyadenosyl radical ( Ado ) generation by homolytic cleavage of a Co–C bond in AdoCbl; (b) substrate activation by Ado H-abstraction from C 1; (c) 1,2-NH 2 migration in substrate’s radical; (d) Ado regeneration; (e) NH 3 elimination from substrate leaving acetaldehyde as final product. Steps a–c have been the subject of both empirical studies and computational modeling performed at the ab initio and DFT levels, with different representations of the protein environment, attempting to explain the observed kinetics by total/ partial protonation or by “ push– pull” catalysis assisted by two amino acid residues. In such models, a drastic reduction of the reactive system and of the influence of the enzyme environment was introduced, the latter mainly due to the lack of detailed data on the 3D structure of EAL (note elucidated until 2007) and the nature of the active site (for which a structure of the substrate- EAL complex in presence of adeninylpentylcobalamin has been very recently reported, shedding light on its structural features). Here we assess at the PCM//UB3LYP/6-31G(d,p) level how using a complete molecular description of both reactants in step b and introducing the polarizing effect of the protein/cofactor environment (mimicked through a continuum model with ε = 10) impacts on the nature and energetics of the intermediate complexes (ICs) and transition states (TSs) under three catalytic scenarios (total, partial, and no protonation). Our results show that a concerted asynchronous mechanism involving distonic radical species is at play, passing through quasi-linear early TSs displaying a C 1–H cleavage slightly more advanced than formation of the new H–C bond in Ado. Whereas the enzyme’s polarizing environment differentially stabilizes the TS lowering the barrier by ∼2.4–2.8 kcal/mol, a reduction of 1.4 kcal/mol in Δ G 298 ≠ is the outcome of substrate’s partial/total protonation, without a clear distinction among them, in opposition to what previously shown based upon more simplified models.

Protection and reactivation of human methylmalonyl-CoA mutase by MMAA protein

Previous studies have reported that some adenosylcobalamin-dependent enzymes suffer inactivation during catalysis due to the oxidation of cobalamin. In addition, the protection or reactivation of their catalytic activities by proteins called “protectases” or reactivases is well known in bacteria. In this study, we examined the influence of human MMAA protein on the kinetics of the reaction catalyzed by methylmalonyl-CoA mutase (MCM) by testing both purified recombinant proteins in vitro. Our results showed that MMAA plays dual roles in MCM activity. When it was added at the beginning of the reaction, it prevents inactivation by guarding MCM. After 60min of reaction, when MCM is inactive, the addition of MMAA increases the enzymatic activity through GTP hydrolysis, indicating reactivation of MCM by exchange of the damaged cofactor. Interaction between MCM and MMAA observed in vitro was confirmed in vivo by yeast two-hybrid system.

Microbe-inspired System Enzymology of Vitamin B<sub>12</sub> Metabolism

Vitamin B₁₂ is produced only by prokaryotes and utilized by animals as an essential micronutrient. Genetic complementation analysis of cell lines from patients indicated that at least eight gene products are involved in intracellular B₁₂ metabolism and utilization. We have investigated bacterial adenosylcobalamin-dependent enzymes and elucidated their structure-based fine mechanisms. They tend to undergo mechanism-based inactivation during catalysis, because they use highly reactive radicals for catalyzing chemically difficult reactions. We have discovered molecular chaperone-like reactivating factors for these enzymes that release a damaged cofactor forming apoenzyme. Methylcobalamin-dependent methionine synthase also undergoes inactivation, because it utilizes cob (I) alamin, a super nucleophile, for catalysis. Methionine synthase reductase is a reactivating partner for this enzyme. Recent studies suggested that activity-maintaining systems for B₁₂ enzymes are present in animal cells as well, and thus hints for designing therapeutic agents for B₁₂-related metabolic disorders might be obtained from the investigations of microbial B₁₂ metabolism.

Hydrogen Tunneling in Adenosylcobalamin-Dependent Glutamate Mutase: Evidence from Intrinsic Kinetic Isotope Effects Measured by Intramolecular Competition

Hydrogen atom transfer reactions between the substrate and coenzyme are key mechanistic features of all adenosylcobalamin-dependent enzymes. For one of these enzymes, glutamate mutase, we have investigated whether hydrogen tunneling makes a significant contribution to the mechanism by examining the temperature dependence of the deuterium kinetic isotope effect associated with the transfer of a hydrogen atom from methylaspartate to the coenzyme. To do this, we designed a novel intramolecular competition experiment that allowed us to measure the intrinsic kinetic isotope effect, even though hydrogen transfer may not be rate-determining. From the Arrhenius plot of the kinetic isotope effect, the ratio of the pre-exponential factors (A(H)/A(D)) was 0.17 +/- 0.04 and the isotope effect on the activation energy [DeltaE(a(D-H))] was 1.94 +/- 0.13 kcal/mol. The results imply that a significant degree of hydrogen tunneling occurs in glutamate mutase, even though the intrinsic kinetic isotope effects are well within the semiclassical limit and are much smaller than those measured for other AdoCbl enzymes and model reactions for which hydrogen tunneling has been implicated.

Continuous Wave Photolysis Magnetic Field Effect Investigations with Free and Protein-Bound Alkylcobalamins

The activation of the Co-C bond in adenosylcobalamin-dependent enzymes generates a singlet-born Co(II)-adenosyl radical pair. Two of the salient questions regarding this process are: (1) What is the origin of the considerable homolysis rate enhancement achieved by this class of enzyme? (2) Are the reaction dynamics of the resultant radical pair sensitive to the application of external magnetic fields? Here, we present continuous wave photolysis magnetic field effect (MFE) data that reveal the ethanolamine ammonia lyase (EAL) active site to be an ideal microreactor in which to observe enhanced magnetic field sensitivity in the adenosylcobalamin radical pair. The observed field dependence is in excellent agreement with that calculated from published hyperfine couplings for the constituent radicals, and the magnitude of the MFE (<18%) is almost identical to that observed in a solvent containing 67% glycerol. Similar augmentation is not observed, however, in the equivalent experiments with EAL-bound methylcobalamin, where all field sensitivity observed in the free cofactor is washed out completely. Parallels are drawn between the latter case and the loss of field sensitivity in the EAL holoenzyme upon substrate binding (Jones et al. J. Am. Chem. Soc. 2007, 129, 15718-15727). Both are attributed to the rapid removal of the alkyl radical immediately after homolysis, such that there is inadequate radical pair recombination for the observation of field effects. Taken together, these results support the notion that rapid radical quenching, through the coupling of homolysis and hydrogen abstraction steps, and subsequent radical pair stabilization make a contribution to the observed rate acceleration of Co-C bond homolysis in adenosylcobalamin-dependent enzymes.

Insights into the mechanisms of adenosylcobalamin (coenzyme B12)-dependent enzymes from rapid chemical quench experiments

Glutamate mutase is one of a group of adenosylcobalamin-dependent enzymes that use free radicals to catalyse unusual and chemically difficult rearrangements involving 1,2-migrations of hydrogen atoms. A key mechanistic feature of these enzymes is the transfer of the migrating hydrogen atom between substrate, coenzyme and product. The present review summarizes recent experiments from my laboratory that have used rapid chemical quench techniques to identify intermediates in the reaction and probe the mechanism of hydrogen transfer through a variety of pre-steady-state kinetic isotope effect measurements.

The S subunit of d-ornithine aminomutase fromClostridium sticklandiiis responsible for the allosteric regulation in d-α-lysine aminomutase

Ornithine and lysine are degraded in quite a similar way in Clostridium sticklandii. Both pathways involve adenosylcobalamin-dependent enzymes, d-ornithine 4,5-aminomutase and lysine 5,6-aminomutase. According to previous reports, lysine 5,6-aminomutase is an ATP-dependent allosteric enzyme with many different activators and inhibitors. However, recent studies indicate that ATP does not have a regulatory effect on the recombinant enzyme. To monitor the activity of lysine aminomutase, a novel capillary electrophoresis-based assay method was developed. The present results demonstrate that the S subunit of d-ornithine aminomutase, OraS, is capable of forming a complex with KamDE of lysine 5,6-aminomutase and restores the enzyme's ATP-dependent allosteric regulation. Not only does ATP lower the K(m) of the KamDE-OraS complex for adenosylcobalamin and pyridoxal phosphate, but also OraS protein alone lowers the K(m) of KamDE for adenosylcobalamin and pyridoxal phosphate. The activity of reconstituted enzyme can also be activated by ammonium ion as reported by Morley and Stadtman.

Synthesis of mono- and di-deuterated (2 S,3 S)-3-methylaspartic acids to facilitate measurement of intrinsic kinetic isotope effects in enzymes

Kinetic isotope effects provide a powerful method to investigate the mechanisms of enzyme-catalyzed reactions, but often other slow steps in the reaction such as substrate binding or product release suppress the isotopically sensitive step. For reactions at methyl groups, this limitation may be overcomed by measuring the isotope effect by an intra-molecular competition experiment. This requires the synthesis of substrates containing regio-specifically mono- or di-deuterated methyl groups. To facilitate the mechanistic investigations of the adenosylcobalamin-dependent enzyme, glutamate mutase, we have developed a synthesis of mono- and di-deuterated (2 S,3 S)-3-methylaspartic acids. Key intermediates are the correspondingly labeled mesaconic acids and their dimethyl esters that potentially provide starting materials for a variety of isotopically labeled molecules.

Evidence for Coupled Motion and Hydrogen Tunneling of the Reaction Catalyzed by Glutamate Mutase

Glutamate mutase is one of a group of adenosylcobalamin-dependent enzymes that catalyze unusual isomerizations that proceed through organic radical intermediates generated by homolytic fission of the coenzyme's unique cobalt-carbon bond. These enzymes are part of a larger family of enzymes that catalyze radical chemistry in which a key step is the abstraction of a hydrogen atom from an otherwise inert substrate. To gain insight into the mechanism of hydrogen transfer, we previously used pre-steady-state, rapid-quench techniques to measure the alpha-secondary tritium kinetic and equilibrium isotope effects associated with the formation of 5'-deoxyadenosine when glutamate mutase was reacted with [5'-(3)H]adenosylcobalamin and L-glutamate. We showed that both the kinetic and equilibrium isotope effects are large and inverse, 0.76 and 0.72, respectively. We have now repeated these measurements using glutamate deuterated in the position of hydrogen abstraction. The effect of introducing a primary deuterium kinetic isotope effect on the hydrogen transfer step is to reduce the magnitude of the secondary kinetic isotope effect to a value close to unity, 1.05 +/- 0.08, whereas the equilibrium isotope effect is unchanged. The significant reduction in the secondary kinetic isotope effect is consistent with motions of the 5'-hydrogen atoms being coupled in the transition state to the motion of the hydrogen undergoing transfer, in a reaction that involves a large degree of quantum tunneling.

Suicide Inactivation in Adenosylcobalamin-Dependent Enzymes

Because organic radicals are highly reactive species, they are traditionally considered to be detrimental to biological systems. Yet intriguingly, adenosylcobalamin (AdoCbl) dependent enzymes exploit the high reactivity of such radicals to facilitate an otherwise difficult rearrangement of adjacent groups.[1,2] A vital component of these reactions is the requirement of 5′-deoxyadenosyl radical (Ado•), derived from the homolytic cleavage of the cobalt–carbon bond of AdoCbl, to mediate two distinct hydrogen atom transfers (Scheme 1). Clearly, the ability of AdoCbl-dependent enzymes to control the specificity of any radical must be met with rigour so as to minimize aberrant side-reactions, a concept often referred to as negative catalysis.[3] However, an emerging theme for these enzymes is the possibility for their irreversible inactivation, resulting from their dependence on radicals for catalysis. One manifestation of this so-called suicide inactivation is unmistakably revealed through the use of suitably chosen AdoCbl and substrate analogues. Analyses of the inactivation products can provide valuable information on

Isotope Effects for Deuterium Transfer between Substrate and Coenzyme in Adenosylcobalamin-Dependent Glutamate Mutase

A key step in the mechanism of all adenosylcobalamin-dependent enzymes is the abstraction of a hydrogen atom from the substrate by a 5'-deoxyadenosyl radical generated by homolytic fission of the coenzyme cobalt-carbon bond. We have investigated the isotope effects associated with this process for glutamate mutase reacting with deuterated glutamate. The kinetics of deuterium incorporation into 5'-deoxyadenosine (5'-dA) during the reaction were followed by rapid chemical quench, using HPLC and electrospray mass spectrometry to analyze the 5'-dA formed. The kinetics of 5'-dA formation are biphasic, comprising a rapid phase k(app) = 37 +/- 3 s(-)(1) and a slower phase k(app) = 0.9 +/- 0.4 s(-)(1). The mass spectral data clearly show that the faster phase is associated with the formation of monodeuterated 5'-dA whereas the slower phase is associated with the incorporation of a second and then a third deuterium into 5'-dA. This observation implies that a large inverse equilibrium secondary isotope effect is associated with the formation of 5'-dA from adenosylcobalamin. The primary deuterium kinetic isotope effects on V and V/K for the formation of 5'-dA were determined from time-based and competition experiments. (D)V = 2.4 +/-0.4 whereas (D)(V/K) = 10 +/- 0.4, implying that an isotopically insensitive step is partially rate-determining. The additional data provided by these experiments cause us to revise our interpretation of earlier UV-visible stopped-flow kinetic measurements of AdoCbl homolysis obtained with deuterated substrates.

Genetic analysis of three genes causing isolated methylmalonic acidemia: identification of 21 novel allelic variants

Isolated methylmalonic aciduria (MMA) is an inborn error of metabolism due to the impaired isomerization of l-methylmalonyl-CoA to succinyl-CoA. This reaction is catalyzed by the mitochondrial protein methylmalonyl-CoA mutase (MCM, EC 5.4.99.2), an adenosylcobalamin-dependent enzyme. Four different forms of isolated MMA have been described: mut MMA associated with defects in the MCM apoenzyme, and phenotypically divided into two subtypes mut − and mut 0 MMA, and three different defects involved in the synthesis of the active form of the cofactor adenosylcobalamin, termed cbl MMA, and classified into three different complementation groups cblA, cblB, and cblH associated with defects in the MMAA and MMAB genes and with an unidentified protein, respectively. In this work we describe the genetic analysis of 25 MMA patients, mainly from Spain. Using biochemical and cellular approaches our patients have been classified, identifying 13 mut MMA, 7 cblA, 2 cblB, and 3 non cblA, non cblB deficient patients. cDNA and genomic DNA sequence analysis of the MUT, MMAA, and MMAB genes have allowed us to identify 27 different changes, 21 novel ones. Among the missense mutations identified in the MUT gene only one, the c.970G > A (p.A324T) variant located in the substrate binding domain is likely a mut − mutation. The remaining missense mutations c.326A > G (p.Q109R), c.983T > C (p.L328P), c.1846C > T (p.R616C), and c.1850T > G (p.L617R) are probably mut 0. In the MMAA patients analyzed, frameshift mutations are prevalent. We have explored the genotype–phenotype correlation for this clinically heterogeneous disease.