In mammals, the two enzymes in trans-sulfuration pathway, cystathionine beta-synthase (CBS) and gamma-lyase (CSE), are believed to be chiefly responsible for hydrogen sulfide (H2S) biogenesis. this study, we report a detailed kinetic analysis of human yeast CBS-catalyzed reactions that result H2S generation. CBS from both organisms shows marked preference generation by beta-replacement cysteine homocysteine. The alternative H2S-generating reactions, i.e. beta-elimination generate serine or...

Although there is a growing recognition of the significance hydrogen sulfide (H2S) as biological signaling molecule involved in vascular and nervous system functions, its biogenesis regulation are poorly understood. It widely assumed that desulfhydration cysteine major source H2S mammals catalyzed by transsulfuration pathway enzymes, cystathionine β-synthase γ-lyase (CSE). In this study, we demonstrate profligacy human CSE results variety reactions generate from homocysteine. The...

MeaB is a recently described P-loop GTPase that plays an auxiliary role in the reaction catalyzed by radical B12 enzyme, methylmalonyl-CoA mutase. Defects human homologue of result methylmalonic aciduria, but this protein coenzyme assimilation and/or utilization not known. Methylmalonyl-CoA mutase catalyzes isomerization to succinyl-CoA uses reactive intermediates are susceptible oxidative inactivation. In study, we have examined influence on kinetics and thermodynamics cofactor binding....

The mechanism by which docking fidelity is achieved for the multitude of cofactor-dependent enzymes poorly understood. In this study, we demonstrate that delivery coenzyme B(12) or 5'-deoxyadenosylcobalamin adenosyltransferase to methylmalonyl-CoA mutase gated a small G protein, MeaB. While GTP-binding energy needed editing function; is, discriminate between active and inactive cofactor forms, chemical GTP hydrolysis required gating transfer. protein chaperone also exerts its function during...

MeaB is an auxiliary protein that supports the function of radical B(12)-dependent enzyme, methylmalonyl-CoA mutase, although its precise role not understood. Mutations in human homolog MeaB, MMAA, lead to methylmalonic aciduria, inborn error metabolism can be fatal. To obtain insights into this recently discovered protein, we have characterized entropic and enthalpic contributions DeltaGdegree (assoc) for complexation (in presence absence nucleotides) with mutase cofactor). The dissociation...

Significance EFA6, cytohesins, and BRAGs activate Arf GTPases in endocytic events. They carry a plasma membrane-binding PH domain tandem with their catalytic Sec7 domain, which is autoinhibitory mediates positive feedback loop cytohesins but not BRAGs, has an as-yet unknown role EFA6 regulation. By reconstituting GDP/GTP exchange on membranes, we find that the of autoinhibitory, supports negative loop. This controlled by interaction Arf6-GTP PH-Ct domains monitors Arf1 Arf6 activation...

Abstract Accumulating evidence suggests that abnormal levels of homocysteine are associated with vascular dysfunctions, cancer cell proliferation and various neurodegenerative diseases. With respect to the latter, a perturbation transition metal homeostasis an inhibition catalase bioactivity have been reported. Herein, we report on some molecular bases for cellular toxicity demonstrate it induces formation sulfcatalase, irreversible inactive state enzyme, without intervention hydrogen...

MeaB is an auxiliary protein that plays a crucial role in the protection and assembly of B(12)-dependent enzyme methylmalonyl-CoA mutase. Impairments human homologue MeaB, MMAA, lead to methylmalonic aciduria, inborn error metabolism. To explore this metallochaperone, its structure was solved nucleotide-free form, as well presence product, GDP. homodimer, with each subunit containing central alpha/beta-core G domain typical GTPase family, alpha-helical extensions at N C termini are not found...

Adenosyltransferases (ATRs) catalyze the synthesis of reactive cobalt-carbon bond found in coenzyme B(12) or 5'-deoxyadenosylcobalamin (AdoCbl), which serves as a cofactor for number isomerases. The reaction involves reductive adenosylation cob(II)alamin an electron delivered by reductase reduces to cob(I)alamin, attacks 5'-carbon ATP form AdoCbl and inorganic triphosphate. Of three classes ATRs nature, PduO type, is also only one mammals, most extensively studied. crystal structures...

The anaerobic ribonucleotide reductase (ARR) from E. coli is the prototype for enzymes that use combination of S-adenosylmethionine (AdoMet) and an iron−sulfur center generating catalytically essential free radicals. ARR a homodimeric α2 protein which acquires glycyl radical during incubation with [4Fe-4S]-containing activating enzyme (β) AdoMet under reducing conditions. Here we show EPR-active S = 1/2 reduced [4Fe-4S]+ cluster competent reductive cleavage, yielding 1 equiv methionine...

In its active form, Escherichia coli class III ribonucleotide reductase homodimer alpha(2) relies on a protein free radical located the Gly(681) residue of alpha polypeptide. The formation glycyl radical, namely, activation enzyme, involves concerted action four components: S-adenosylmethionine (AdoMet), dithiothreitol (DTT), an Fe-S called beta or "activase", and reducing system consisting NADPH, NADPH:flavodoxin oxidoreductase, flavodoxin (fldx). It has been proposed that reductant serves...

Coenzyme B(12) is used by two highly similar radical enzymes, which catalyze carbon skeleton rearrangements, methylmalonyl-CoA mutase and isobutyryl-CoA (ICM). ICM catalyzes the reversible interconversion of n-butyryl-CoA exists as a heterotetramer. In this study, we have identified >70 bacterial proteins, represent fusions between subunits P-loop GTPase are currently misannotated mutases. We designate fusion protein IcmF (isobutyryl-CoA fused). All IcmFs composed following three domains:...

Persulfides are an emerging class of cysteine oxidative post-translational modification. They react with the bioconjugation reagents bicyclo[6.1.0]nonynes (BCNs) to engender thioethers and/or disulfides. This new reactivity BCNs a biologically important redox-signaling species efficiently interferes recent usage strained cycloalkynes specifically trap protein sulfenic acids.

Persulfides reduce both met- and ferryl-oxidized forms of myoglobin, coordinate to<italic>N</italic>-acetylated microperoxidase-11.

Hydrogen sulfide (H2S) is a gaseous signaling molecule that participates in various functions health and diseases. The tetrameric cystathionine γ-lyase (CSE) contributes to H2S biogenesis several investigations provide evidence on the pharmacological modulation of CSE as potential target for treatment multitude conditions. D-penicillamine (D-pen) has recently been reported selectively impede CSE-catalyzed production but molecular bases such inhibitory effect have not investigated. In this...

Small GTP-binding proteins of the Arf family (Arf GTPases) interact with multiple cellular partners and membranes to regulate intracellular traffic organelle structure. Understanding underlying molecular mechanisms requires in vitro biochemical assays test for regulations functions. Such should use their form, which carry a myristoyl lipid attached N-terminus. N-myristoylation recombinant GTPases can be achieved by co-expression E. coli eukaryotic N-myristoyl transferase. However, purifying...

The complex chemistry taking place between the messenger 8-nitro-cGMP and gaseous transmitter H₂S is governed by dioxygen thiols.

Methylmalonyl-CoA mutase catalyzes the adenosylcobalamin-dependent rearrangement of (2R)-methylmalonyl-CoA to succinyl-CoA. The crystal structure enzyme reveals that Y243 is in van der Waals contact with methyl group substrate and suggests a possible role for it stereochemical control reaction. This hypothesis was tested by designing molecular hole replacing phenolic side chain alanine. Y243A mutation lowered catalytic efficiency >(4 × 104)-fold compared wild-type enzyme, KMapp cofactor...