Clamping down on weak terminal base pairs: oligonucleotides with molecular caps as fidelity-enhancing elements at the 5'- and 3'-terminal residues

The base-pairing fidelity of oligonucleotides depends on the identity of the nucleobases involved and the position of matched or mismatched base pairs in the duplex. Nucleobases forming weak base pairs, as well as a terminal position favor mispairing. We have searched for 5′-appended acylamido caps that enhance the stability and base-pairing fidelity of oligonucleotides with a 5′-terminal 2′-deoxyadenosine residue using combinatorial synthesis and MALDI-monitored nuclease selections. This provided the residue of 4-(pyren-1-yl)butyric acid as a lead. Lead optimization gave (S)-N-(pyren-1-ylmethyl)pyrrolidine-3-phosphate as a cap that increases duplex stability and base-pairing fidelity. For the duplex of 5′-AGGTTGAC-3′ with its fully complementary target, this cap gives an increase in the UV melting point T_m of +10.9°C. The T_m is 6.3–8.3°C lower when a mismatched nucleobase faces the 5′-terminal dA residue. The optimized cap can be introduced via automated DNA synthesis. It was combined with an anthraquinone carboxylic acid residue as a cap for the 3′-terminal residue. A doubly capped dodecamer thus prepared gives a melting point decrease for double-terminal mismatches that is 5.7–5.9°C greater than that for the unmodified control duplex.     

Purification and N-terminal analyses of algal biliproteins

1. R-, B- and C-phycoerythrins and R- and C-phycocyanins were isolated and purified on a preparative scale by calcium phosphate chromatography, ammonium sulphate fractionation and crystallization. 2. The N-terminal residues of these biliproteins were analysed. Methionine is the only N-terminal residue of all the phycoerythrins, there being about 14 N-terminal residues per molecule of R- and B-phycoerythrins (mol.wt. 290000) and about 8 per molecule of C-phycoerythrin (mol.wt. 226000). Threonine (1 residue) is N-terminal in C-phycocyanin (mol.wt. 138000), and both threonine (about 1·3 residues) and methionine (5 residues) are N-terminal in R-phycocyanin (mol.wt. 273000). 3. Results suggest that the apoproteins of the various phycoerythrins are closely related, whereas C-phycocyanin has quite a different gross structure, and that R-phycocyanin contains two types of sub-unit, one related to C-phycocyanin and the other to the phycoerythrins.     

A Short-lived Intermediate Form in the in Vivo Conversion of Protochlorophyllide 650 to Chlorophyllide 684

When dark-grown leaves of Phaseolus vulgaris, Hordeum vulgare, Zea mays and Pisum sativum were irradiated for 3 sec at 2° the first product of protochlorophyllide 650 conversion had an absorption maximum at 678 nm. This form was then converted in a dark reaction to chlorophyllide 684, the form generally observed and regarded as the in vivo product of the photoreaction. The dark conversion at 2° was complete in 6 to 10 min in the various plants. The time course of the dark reaction was followed at 690 nm near the maximum of the difference spectrum for the conversion. There was a constant relationship between the initial amount of chlorophyllide 678 and the final amount of chlorophyllide 684. The rates of the dark reaction at 2° varied 3-fold among the plants treated. The reaction was not first order. At 25° the reaction followed at 690 nm was complete in 20 to 60 sec. Q₁₀'s varied from 2.8 to 3.7 between 2° and 25°. Phytochrome absorbancy changes were shown to be too low to interfere with these measurements except in pea leaves. In a subsequent stage of greening newly regenerated protochlorophyllide went through the same sequence upon photoconversion. Chlorophyllide 678 probably corresponds to the product formed in vitro from the protochlorophyllide holochrome. The dark reaction appears to represent the first interaction between the photoconverted holochrome and other elements of the proplastid. The lack of this dark reaction could also account for the spectral properties of certain albino mutants.     

Purification, Characterization, Gene Cloning, Sequencing, and Overexpression of Aminopeptidase N from Streptococcus thermophilus A

The general aminopeptidase PepN from Streptococcus thermophilus A was purified to protein homogeneity by hydroxyapatite, anion-exchange, and gel filtration chromatographies. The PepN enzyme was estimated to be a monomer of 95 kDa, with maximal activity on N-Lys–7-amino-4-methylcoumarin at pH 7 and 37°C. It was strongly inhibited by metal chelating agents, suggesting that it is a metallopeptidase. The activity was greatly restored by the bivalent cations Co²⁺, Zn²⁺, and Mn²⁺. Except for proline, glycine, and acidic amino acid residues, PepN has a broad specificity on the N-terminal amino acid of small peptides, but no significant endopeptidase activity has been detected. The N-terminal and short internal amino acid sequences of purified PepN were determined. By using synthetic primers and a battery of PCR techniques, the pepN gene was amplified, subcloned, and further sequenced, revealing an open reading frame of 2,541 nucleotides encoding a protein of 847 amino acids with a molecular weight of 96,252. Amino acid sequence analysis of the pepN gene translation product shows high homology with other PepN enzymes from lactic acid bacteria and exhibits the signature sequence of the zinc metallopeptidase family. The pepN gene was cloned in a T7 promoter-based expression plasmid and the 452-fold overproduced PepN enzyme was purified to homogeneity from the periplasmic extract of the host Escherichia coli strain. The overproduced enzyme showed the same catalytic characteristics as the wild-type enzyme.     

Mineralization of Carbofuran by a Soil Bacterium

A bacterium, tentatively identified as an Arthrobacter sp., was isolated from flooded soil that was incubated at 35°C and repeatedly treated with carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl N-methylcarbamate). This bacterium exhibited an exceptional capacity to completely mineralize the ring-labeled ¹⁴C in carbofuran to ¹⁴CO₂ within 72 to 120 h in a mineral salts medium as a sole source of carbon and nitrogen under aerobic conditions. Mineralization was more rapid at 35°C than at 20°C. No degradation of carbofuran occurred even after prolonged incubation under anaerobic conditions. The predicted metabolites of carbofuran, 7-phenol (2,3-dihydro-2,2-dimethyl-7-benzofuranol) and 3-hydroxycarbofuran, were also metabolized rapidly. 7-Phenol, although formed during carbofuran degradation, never accumulated in large amounts, evidently because of its further metabolism through ring cleavage. The bacterium readily hydrolyzed carbaryl (1-naphthyl N-methylcarbamate), but its hydrolysis product, 1-naphthol, resisted further degradation by this bacterium.     

Activity of ribulose 1,5-bisphosphate carboxylase oxygenase as a function of storage conditions

Optimal storage conditions to retain ribulose 1, 5-bisphosphate carboxylase/oxygenase (Rubisco) activity were investigated. The soluble spinach (Spinacia oleracea) enzyme was pretreated with its activators, Mg²⁺ and HCO₃⁻, and then stored for up to 30 days at 4 or −18°C or in liquid N₂. Cold inactivation and conformational changes were suggested to be involved during Rubisco storage in the cold, leading to its inactivation. Pretreatment of the enzyme with Mg²⁺ and CO₂ and subsequent storage at either 4°C or in liquid N₂ or flushing the samples with N₂ and rapid freezing and storage in liquid N₂ are recommended as storage procedures. These storage treatments will prevent inactivation, so that full original specific activity will be preserved.     

Structural and dielectric properties of synthetic glycolipids in mixtures with water

Three synthetically produced glycolipids, N-(β-D-glucopyranosyl)-N-octadecyl-stearoylamide (OSGA), N-(β-D-glucopyranosyl-N-octadecyl-oleoylamide (OOGA), N-(β-D-galactopyranosyl)-N-octadecyl-lauroylamide (OLGA) have been studied in different mixtures with water by x-ray diffraction and dielectric measurements with microwaves at 9.4 GHz. The measurements were performed in the temperature range -50-70°C. X-Ray diffraction revealed a direct L_β' → H transition at 20°C, 60°C, and 45°C depending on the glycolipid species but nearly not on the water content. The hexagonal phases are saturated at a water content of ≈20 wt%. The lamellar phase absorbs even less water (< 10 wt%). The dielectric data show that in the H phase the binding of water is stronger than in the L_β' phase. In the temperature range below 0°C, OSGA and OOGA show a “subzero transition” due to the freeze-out of water in a separate ice phase. This transition can be seen in an abrupt decrease of the dielectric function because the dielectric response of ice is much smaller at microwave frequencies. OLGA does not show the subzero transition but an additional transition, hexagonal → distorted hexagonal at 60°C.     

Stimulation of human 8-oxoguanine-DNA glycosylase by AP-endonuclease: potential coordination of the initial steps in base excision repair

8-Oxoguanine-DNA glycosylase 1 (OGG1), with intrinsic AP lyase activity, is the major enzyme for repairing 7,8-dihydro-8-oxoguanine (8-oxoG), a critical mutagenic DNA lesion induced by reactive oxygen species. Human OGG1 excised the damaged base from an 8-oxoG·C-containing duplex oligo with a very low apparent k_cat of 0.1 min^–1 at 37°C and cleaved abasic (AP) sites at half the rate, thus leaving abasic sites as the major product. Excision of 8-oxoG by OGG1 alone did not follow Michaelis–Menten kinetics. However, in the presence of a comparable amount of human AP endonuclease (APE1) the specific activity of OGG1 was increased ~5-fold and Michaelis–Menten kinetics were observed. Inactive APE1, at a higher molar ratio, and a bacterial APE (Nfo) similarly enhanced OGG1 activity. The affinity of OGG1 for its product AP·C pair (K_d ~ 2.8 nM) was substantially higher than for its substrate 8-oxoG·C pair (K_d ~ 23.4 nM) and the affinity for its final β-elimination product was much lower (K_d ~ 233 nM). These data, as well as single burst kinetics studies, indicate that the enzyme remains tightly bound to its AP product following base excision and that APE1 prevents its reassociation with its product, thus enhancing OGG1 turnover. These results suggest coordinated functions of OGG1 and APE1, and possibly other enzymes, in the DNA base excision repair pathway.     

Purification and Characterization of an Arginine Aminopeptidase from Lactobacillus sakei

An arginine aminopeptidase (EC 3.4.11.6) that exclusively hydrolyzes basic amino acids from the amino (N) termini of peptide substrates has been purified from Lactobacillus sakei. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included hydrophobic interaction, gel filtration, and anion-exchange chromatography. This procedure resulted in a recovery rate of 4.2% and a 500-fold increase in specific activity. The aminopeptidase appeared to be a trimeric enzyme with a molecular mass of 180 kDa. The activity was optimal at pH 5.0 and 37°C. The enzyme was inhibited by sulfhydryl group reagents and several divalent cations (Cu²⁺, Hg²⁺, and Zn²⁺) but was activated by reducing agents, metal-chelating agents, and sodium chloride. The enzyme showed a preference for arginine at the N termini of aminoacyl derivatives and peptides. The K_m values for Arg-7-amido-4-methylcoumarin (AMC) and Lys-AMC were 15.9 and 26.0 μM, respectively. The nature of the amino acid residue at the C terminus of dipeptides has an effect on hydrolysis rates. The activity was maximal toward dipeptides with Arg, Lys, or Ala as the C-terminal residue. The properties of the purified enzyme, its potential function in the release of arginine, and its further metabolism are discussed because, as a whole, it could constitute a survival mechanism for L. sakei in the meat environment.     

Formation of 2'-deoxyoxanosine from 2'-deoxyguanosine and nitrous acid: mechanism and intermediates

The reaction mechanism for the formation of 2′-deoxyoxanosine from 2′-deoxyguanosine by nitrous acid was explored using methyl derivatives of guanosine and an isolated intermediate of the reaction. When 1-methylguanosine was incubated with NaNO₂ under acidic conditions, N⁵-methyloxanosine and 1-methylxanthosine were generated, whereas the same treatment of N²,N²-dimethylguanosine generated no product. In a similar experiment without NO₂^–, participation of a Dimroth rearrangement was ruled out. In the guanosine–HNO₂ reaction system, an intermediate with a half-life of 5.6 min (pH 7.0, 20°C) was isolated and tentatively identified as a diazoate derivative of guanosine. The diazoate intermediate was converted into oxanosine and xanthosine at a molar ratio (oxanosine:xanthosine) of 0.26 at pH 7.0 and 20°C. The ratio was not affected by the incubation pH between 2 and 10, but increased linearly with temperature from 0.22 (0°C) to 0.32 (50°C). The addition of acetone also increased the ratio up to 0.85 (98% acetone). Based on these results, a con-ceivable pathway for the formation of 2′-deoxyoxanosine from 2′-deoxyguanosine by HNO₂ is proposed.     

Purification and Characterization of Leu-Proteinase, the Leucine Specific Serine Proteinase from Spinach (Spinacia oleracea L.) Leaves

The leucine specific serine proteinase present in the soluble fraction of leaves from Spinacia oleracea L. (called Leu-proteinase) has been purified by acetone precipitation and a combination of gel-filtration, ion exchange, and adsorption chromatography. This enzyme shows a molecular weight of 60,000 ± 3,000 daltons, an isoelectric point of 4.8 ± 0.1, and a relative electrophoretic mobility of 0.58 ± 0.03. The Leu-proteinase catalyzed hydrolysis of p-nitroanilides of N-α-substituted(-l-)amino acids as well as of chromogenic macromolecular substrates has been investigated between pH 5 and 10 at 23 ± 0.5°C and I = 0.1 molar. The enzyme activity is characterized by a bell-shaped profile with an optimum pH value around 7.5, reflecting the acid-base equilibrium of groups with pK_a values of 6.8 ± 0.1 and 8.2 ± 0.1 (possibly the histidyl residue present at the active site of the enzyme and the N-terminus group). Among the substrates considered, N-α-benzoyl-l-leucine p-nitroanilide shows the most favorable catalytic parameters and allows to determine an enzyme concentration as low as 1 × 10⁻⁹ molar. In agreement with the enzyme specificity, only N-α-tosyl-l-leucine chloromethyl ketone, di-isopropyl fluorophosphate and phenylmethylsulfonyl fluoride, among compounds considered specific for serine enzymes, strongly inhibit the Leu-proteinase. Accordingly, the enzyme activity is insensitive to cations, chelating agents, sulfydryl group reagents, and activators.     

Indirect Measurement of the Lag Time Distribution of Single Cells of Listeria innocua in Food

The distribution of log counts at a given time during the exponential growth phase of Listeria innocua measured in food samples inoculated with one cell each was applied to estimate the distribution of the single-cell lag times. Three replicate experiments in broth showed that the distribution of the log counts is a linear mapping of the distribution of the detection times measured by optical density. The detection time distribution reflects the lag time distribution but is shifted in time. The log count distribution was applied to estimate the distributions of the lag times in a liquid dairy product and in liver paté after different heat treatments. Two batches of ca. 100 samples of the dairy product were inoculated and heated at 55°C for 45 min or at 62°C for 2 min, and an unheated batch was incubated at 4°C. The final concentration of surviving bacteria was ca. 1 cell per sample. The unheated cells showed the shortest lag times with the smallest variance. The mean and the variance of the lag times of the surviving cells at 62°C were greater than those of the cells treated at 55°C. Three batches of paté samples were heated at 55°C for 25 min, 62°C for 81 s, or 65°C for 20 s. A control batch was inoculated but not heated. All paté samples were incubated at 15°C. The distribution of the lag times of the cells heated at 55°C was not significantly different from that of the unheated cells. However, at the higher temperatures, 62°C and 65°C, the lag duration was longer and its variance greater.     

Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic isotopes are typically incorporated metabolically to improve the sensitivity and allow for sequential resonance assignment. Reductive ¹³C-methylation is an alternative labeling method for proteins that are not amenable to bacterial host over-expression, the most common method of isotope incorporation. Reductive ¹³C-methylation is a chemical reaction performed under mild conditions that modifies a protein''s primary amino groups (lysine ε-amino groups and the N-terminal α-amino group) to ¹³C-dimethylamino groups. The structure and function of most proteins are not altered by the modification, making it a viable alternative to metabolic labeling. Because reductive ¹³C-methylation adds sparse, isotopic labels, traditional methods of assigning the NMR signals are not applicable. An alternative assignment method using mass spectrometry (MS) to aid in the assignment of protein ¹³C-dimethylamine NMR signals has been developed. The method relies on partial and different amounts of ¹³C-labeling at each primary amino group. One limitation of the method arises when the protein''s N-terminal residue is a lysine because the α- and ε-dimethylamino groups of Lys1 cannot be individually measured with MS. To circumvent this limitation, two methods are described to identify the NMR resonance of the ¹³C-dimethylamines associated with both the N-terminal α-amine and the side chain ε-amine. The NMR signals of the N-terminal α-dimethylamine and the side chain ε-dimethylamine of hen egg white lysozyme, Lys1, are identified in ¹H-¹³C heteronuclear single-quantum coherence spectra.     

The thermal polymerization of orosomucoid

1. Orosomucoid was prepared from the urine of a nephrotic patient and polymerized by heating it in a range of salt concentrations at pH4·1. 2. Heating at low ionic strengths produced a `chain' polymer of indefinite length but having the same width as the diameter of the monomer (5·0nm.). Similar treatment in high ionic strengths also produced a spherical (`ball') polymer of limited diameter (14·8nm.). 3. The size and shape of both polymers were determined from ultra-centrifuge, gel-filtration and electron-microscope results. The results suggest that eight monomer units condense to form the ball polymer. 4. Heating orosomucoid at pH1·8 hydrolysed the N-acetylneuraminic acid off the molecule; only chains could then be formed, even in high ionic strengths. 5. Both polymers were stable under normal conditions but could be depolymerized in 3m-guanidine hydrochloride. The monomer could be repolymerized on heating: the `chain monomer' only formed chains at all ionic strengths, but the `ball monomer' was indistinguishable from the original monomer in its immunological properties and polymerization reaction.     

Studies on the mucin derived from human colloid breast carcinoma

1. A non-diffusible mucoid, showing a single peak in the ultracentrifuge, was isolated from human colloid breast carcinoma by treatment with trypsin and pepsin. The material contained threonine, leucine (isoleucine), valine, proline, glycine and glutamic acid in the approximate molar proportions 5:1:1:2:1:1. Smaller amounts of aspartic acid and serine were also found. For each 5 threonine residues, 6 N-acetylgalactosamine and 3–4 galactose residues were present. 2. The mucoid possessed reducing properties by the Park & Johnson (1949) procedure; these were attributable to the action of mild alkali, as employed in this procedure. Mild alkaline treatment by the Aminoff, Morgan & Watkins (1952) procedure gave rise to a diffusible N-acetylgalactosamine chromophore that gave an enhanced colour with Ehrlich's reagent. That galactosyl-(1→3)-N-acetylgalactosamine residues were liberated was supported by periodate studies. 3. Alkaline liberation of hexosamine residues was accompanied by a specific destruction of threonine. After 40 min. at 100° in 0·18 n-lithium hydroxide, both moieties had almost completely disappeared from the ninhydrin-positive components formed on subsequent acid hydrolysis. Glycine and α-oxobutyric acid were present in the acid hydrolysate, showing that both possible pathways of a β-elimination reaction were involved. Formation of diffusible peptide on very mild alkaline treatment was attributable to the rupture of the original peptide core, necessitated by the second of these two pathways. 4. Hydroxamate formation on treatment with hydroxylamine showed the presence of carbohydrate linkage to glutamic acid or aspartic acid residues or both. This could account for the single N-acetylgalactosamine residue not linked to threonine. 5. The native mucin contained sialic acid, which was cleaved by the acid environment used in the treatment with pepsin. A statistical model of the mucin would require each prosthetic group to be linked, via N-acetylgalactosamine, to threonine, which would occupy every alternate position among the amino acids in the peptide core.     

An Extra Peptide within the Catalytic Module of a β-Agarase Affects the Agarose Degradation Pattern

Agarase hydrolyzes agarose into a series of oligosaccharides with repeating disaccharide units. The glycoside hydrolase (GH) module of agarase is known to be responsible for its catalytic activity. However, variations in the composition of the GH module and its effects on enzymatic functions have been minimally elucidated. The agaG4 gene, cloned from the genome of the agarolytic Flammeovirga strain MY04, encodes a 503-amino acid protein, AgaG4. Compared with elucidated agarases, AgaG4 contains an extra peptide (Asn²⁴⁶–Gly³⁰²) within its GH module. Heterologously expressed AgaG4 (recombinant AgaG4; rAgaG4) was determined to be an endo-type β-agarase. The protein degraded agarose into neoagarotetraose and neoagarohexaose at a final molar ratio of 1.5:1. Neoagarooctaose was the smallest substrate for rAgaG4, whereas neoagarotetraose was the minimal degradation product. Removing the extra fragment from the GH module led to the inability of the mutant (rAgaG4-T57) to degrade neoagarooctaose, and the final degradation products of agarose by the truncated protein were neoagarotetraose, neoagarohexaose, and neoagarooctaose at a final molar ratio of 2.7:2.8:1. The optimal temperature for agarose degradation also decreased to 40 °C for this mutant. Bioinformatic analysis suggested that tyrosine 276 within the extra fragment was a candidate active site residue for the enzymatic activity. Site-swapping experiments of Tyr²⁷⁶ to 19 various other amino acids demonstrated that the characteristics of this residue were crucial for the AgaG4 degradation of agarose and the cleavage pattern of substrate.     

The tissue content and turnover rates of intermediates in the biosynthesis of glycosaminoglycans in young rat skin

1. The tissue contents of hexose monophosphate, N-acetylglucosamine 6-phosphate, UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and UDP-glucuronic acid were determined in the skin of young rats less than 1 day post partum. Tissue-space determinations were used to calculate their average cellular concentrations. 2. The incorporation of [U-¹⁴C]-glucose into the intermediates was recorded with time and their rates of turnover were calculated. The results demonstrated product–precursor relationships along the pathway of hexosamine synthesis and that of hexuronic acid synthesis. The rates of synthesis of UDP-N-acetylhexosamine and UDP-glucuronic acid were 1·5±0·3 and 0·24±0·03mμmoles/min./g. of tissue respectively. These results indicated the average turnover time of the total tissue glycosaminoglycans to be about 5 days.     

A fluorimetric method for the determination of tryptophan in animal tissues

Tryptophan, tryptamine and peptides containing N-terminal tryptophan give two highly fluorescent products on treatment with dithiothreitol and acid ninhydrin reagent 1 or 2. The first fluorescent product (product A) gives an emission at 500nm on activation at 390–400nm and is stable for 20min. The second product (product B), which gives an emission at 530nm on activation at 470nm, is detectable within 1h after the reaction. It gives almost maximum intensity in 4h and is stable for at least 48h. Except lysine, which in equimolar amounts gives less than 1% of a product similar to product B, no other naturally occurring amino compounds give fluorescent products. A procedure is given for the determination of 0.05–34nmol of tryptophan in tissue extracts. By using this procedure rat brain was found to contain 17.56±0.76 (s.e.m.) nmol/g wet wt.     

Genetic Analysis of a Baculovirus, Autographa californica Nuclear Polyhedrosis Virus I. Isolation of Temperature-Sensitive Mutants and Assortment into Complementation Groups

Temperature-sensitive (ts) mutants were isolated from the baculovirus Autographa californica (alfalfa looper) MNPV, grown in Spodoptera frugiperda (fall armyworm) cells in the presence of N-methyl-N′-nitro-N-nitrosoguanidine. Of 567 plaque isolates screened, 27 were temperature sensitive (ts), representing a mutation frequency of 4.8%. Ten ts mutants were studied in detail: six failed to yield nonoccluded virus at 33°C (NOV mutants), whereas the other four produced nonoccluded virus but were restricted in formation of polyhedra at 33°C (Poly mutants). One of the six NOV mutants failed to synthesize viral DNA. Reversion and leak frequencies were determined, and the mutants were assorted into complementation groups based on the yield of polyhedrin synthesis in cells coinfected with pairs of mutants at 33°C, as measured by radioimmunoassay. For NOV mutants, complementation indexes were also based on virus yield and were consistent with those based on polyhedrin synthesis. Nine mutants were assorted into five complementation groups. One mutant remained unclassified.     

The Reaction Mechanism of Methyl-Coenzyme M Reductase: HOW AN ENZYME ENFORCES STRICT BINDING ORDER*

Methyl-coenzyme M reductase (MCR) is a nickel tetrahydrocorphinoid (coenzyme F430) containing enzyme involved in the biological synthesis and anaerobic oxidation of methane. MCR catalyzes the conversion of methyl-2-mercaptoethanesulfonate (methyl-SCoM) and N-7-mercaptoheptanoylthreonine phosphate (CoB₇SH) to CH₄ and the mixed disulfide CoBS-SCoM. In this study, the reaction of MCR from Methanothermobacter marburgensis, with its native substrates was investigated using static binding, chemical quench, and stopped-flow techniques. Rate constants were measured for each step in this strictly ordered ternary complex catalytic mechanism. Surprisingly, in the absence of the other substrate, MCR can bind either substrate; however, only one binary complex (MCR·methyl-SCoM) is productive whereas the other (MCR·CoB₇SH) is inhibitory. Moreover, the kinetic data demonstrate that binding of methyl-SCoM to the inhibitory MCR·CoB₇SH complex is highly disfavored (K_d = 56 mm). However, binding of CoB₇SH to the productive MCR·methyl-SCoM complex to form the active ternary complex (CoB₇SH·MCR(Ni^I)·CH₃SCoM) is highly favored (K_d = 79 μm). Only then can the chemical reaction occur (k_obs = 20 s⁻¹ at 25 °C), leading to rapid formation and dissociation of CH₄ leaving the binary product complex (MCR(Ni^II)·CoB₇S⁻·SCoM), which undergoes electron transfer to regenerate Ni(I) and the final product CoBS-SCoM. This first rapid kinetics study of MCR with its natural substrates describes how an enzyme can enforce a strictly ordered ternary complex mechanism and serves as a template for identification of the reaction intermediates.