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The method of hydrogen exchange mass spectrometry is used to map peptides whose motions are correlated with transmission of intrasteric inhibition and allosteric activation in Cystathionine beta-synthase enzyme, which plays a role in heart disease in humans. The data collected reveal an excellent correlation between kinetically and conformationally distinguishable states of the enzyme and a pathogenic regulatory domain mutant is reported and a structural model of the enzyme is built.

Many organic cofactors are both rare and reactive. They are usually in low abundance, which poses problems for efficient collision-based targeting to dependent enzymes, whereas their reactivity is problematic for side reactions. Sequestration and escorted delivery presents one solution to this conundrum, but such porters, if they exist, are mostly unknown. In humans, the mitochondrial enzyme methylmalonyl-coenzyme A mutase uses coenzyme [B.sub.12] (adenosylcobalamin) but would be inactive if bound to the cofactor precursor that is delivered to the mitochondrion. Adenosyltransferase converts cob(II)alamin to coenzyme [B.sub.12]. Based on kinetic evidence for interaction between the two enzymes, the 40-fold greater affinity for coenzyme [B.sub.12] and the higher coordination number for cobalt in the mutase, we propose that the adenosyltransferase is a dual-function protein: an enzyme that synthesizes coenzyme [B.sub.12] and a chaperone that delivers it.

Electron paramagnetic resonance spectroscopy was used to investigate the interaction between diol dehydratase and adenosylcobalamin and the function of 5,6-dimethylbenzimidazole in this interaction. Results reveal that 5,6-dimethylbenzimidazole coordinates the interaction between the cobalt atom of adenosylcobalamin and diol dehydratase. The utility of a adenosylcobalamin analog in the investigation of the function of 5,6-dimethylbenzimidazole is discussed.

The preparation and catalytic properties of mutant diol dehydratases in which His[alpha]143 is substituted with another amino acid residue are described. The holoenzyme of H[alpha]143Q has undergone irreversible inactivation by [O.sub.2] in the absence of substrate at a much lower rate than the wild type.

A study is conducted, in which several deuterated and (super 13)C-labeled 1,2-propanediols are synthesized and the EPR spectra of holodiol dehydratase reacting with them is measured. By comparison between the spectra with labeled and unlabeled substrates, the organic radical intermediate in the diol dehydratase reaction is identified as the 1,2-propanediol-1-yl-radical, a C1-centered substrate-derived radical.

Spectroscopic analysis of human adenosyltransferase, the locus of the cobalamin (Cbl)B set of patient mutations, reveals an unusual coordination environment for the bound B(sub 12) cofactor. It attains the mirror 'base-off' conformation of cobalmin in the enzyme that synthesis the active coenzyme form and the enzyme that utilizes it.

Research reveals that the metabolism of cyclopropanecarboxylate in Rhodococcus rhodochrous follows an oxidative degradation through cyclopropanecarboxyl-CoA and 3-hydroxybutyryl-CoA. Data indicate that the enzymes required for catalyzing formation and ring opening of cyclopropanecarboxyl-CoA are inducible while the other catabolic enzymes are constitutive.

Spectroscopic evidence for the formation of an unprecedented four-coordinate Co(sub 2+)cobalamin intermediate during adenosylcobalamin (AdoCbl) biosynthesis catalyzed by human adenosyltransferase (hATR) is presented. Results indicate that in the absence of the cosubstrate ATP, the interaction between Co(sub 2+)cobalamin and hATR promotes partial conversion of the cofactor to the base-off form in which a water molecule occupies the lower axial position.

Coenzyme B12 dependent diol dehydratase undergoes mechanism-based inactivation by glycerol, accompanying the irreversible cleavage of the coenzyme Co-C bond. Bachovchin et al. [ Biochemistry 16, 1082-1092 (1977)] reported that glycerol bound in the GS conformation, in which the pro-S-CH2OH group is oriented to the hydrogen-abstracting site, primarily contributes to the inactivation reaction. To understand the mechanism of inactivation by glycerol, we analyzed the X-ray structure of diol dehydratase complexed with cyanocobalamin and glycerol. Glycerol is bound to the active site preferentially in the same conformation as that of ( S)-1,2-propanediol, i.e. in the GS conformation, with its 3-OH group hydrogen bonded to Serα301, but not to nearby Glnα336. kinact of the Sα301A, Qα336A and Sα301A/Qα336A mutants with glycerol was much smaller than that of the wild-type enzyme. kcat/ kinact showed that the Sα301A and Qα336A mutants are substantially more resistant to glycerol inactivation than the wild-type enzyme, suggesting that Serα301 and Glnα336 are directly or indirectly involved in the inactivation. The degree of preference for ( S)-1,2-propanediol decreased on these mutations. The substrate activities towards longer chain 1,2-diols significantly increased on the Sα301A/Qα336A double mutation, probably because these amino acid substitutions yield more space for accommodating a longer alkyl group on C3 of 1,2-diols. Database Structural data are available in the Protein Data Bank under the accession number . Structured digital abstract [ABSTRACT FROM AUTHOR]

Many organic cofactors are both rare and reactive. They are usually in low abundance, which poses problems for efficient collision-based targeting to dependent enzymes, whereas their reactivity is problematic for side reactions. Sequestration and escorted delivery presents one solution to this conundrum, but such porters, if they exist, are mostly unknown. In humans, the mitochondrial enzyme methylmalonyl-coenzyme A mutase uses coenzyme B12 (adenosylcobalamin) but would be inactive if bound to the cofactor precursor that is delivered to the mitochondrion. Adenosyltransferase converts cob(II)alamin to coenzyme B12. Based on kinetic evidence for interaction between the two enzymes, the 40-fold greater affinity for coenzyme B12 and the higher coordination number for cobalt in the mutase, we propose that the adenosyltransferase is a dual-function protein: an enzyme that synthesizes coenzyme B12 and a chaperone that delivers it. [Copyright &y& Elsevier]

Recombinant glycerol dehydratase of Klebsiella pneumoniae was purified to homogeneity. The subunit composition of the enzyme was most probably α2 β2 γ2 . When (R )- and (S )-propane-1,2-diols were used independently as substrates, the rate with the (R )-enantiomer was 2.5 times faster than that with the (S )-isomer. In contrast to diol dehydratase, an isofunctional enzyme, the affinity of the enzyme for the (S )-isomer was essentially the same or only slightly higher than that for the (R )-isomer (K m(R ) /K m(S ) = 1.5). The crystal structure of glycerol dehydratase in complex with cyanocobalamin and propane-1,2-diol was determined at 2.1 Å resolution. The enzyme exists as a dimer of the αβγ heterotrimer. Cobalamin is bound at the interface between the α and β subunits in the so-called ‘base-on’ mode with 5,6-dimethylbenzimidazole of the nucleotide moiety coordinating to the cobalt atom. The electron density of the cyano group was almost unobservable, suggesting that the cyanocobalamin was reduced to cob(II)alamin by X-ray irradiation. The active site is in a (β/α)8 barrel that was formed by a central region of the α subunit. The substrate propane-1,2-diol and essential cofactor K+ are bound inside the (β/α)8 barrel above the corrin ring of cobalamin. K+ is hepta-coordinated by the two hydroxyls of the substrate and five oxygen atoms from the active-site residues. These structural features are quite similar to those of diol dehydratase. A closer contact between the α and β subunits in glycerol dehydratase may be reminiscent of the higher affinity of the enzyme for adenosylcobalamin than that of diol dehydratase. Although racemic propane-1,2-diol was used for crystallization, the substrate bound to glycerol dehydratase was assigned to the (R )-isomer. This is in clear... [ABSTRACT FROM AUTHOR]

The roles of the D-ribosyl moiety and the bulky axial ligand of the nucleotide loop of adenosylcobalamin in coenzymic function have been investigated using two series of coenzyme analogs bearing various artificial bases. The 2-methylbenzimidazolyl trimethylene analog that exists exclusively in the base-off form was a totally inactive coenzyme for diol dehydratase and served as a competitive inhibitor. The benzimidazolyl trimethylene analog and the benzimidazolylcobamide coenzyme were highly active for diol dehydratase and ethanolamine ammonia-lyase. The imidazolylcobamide coenzyme was 59 and 9% as active as the normal coenzyme for diol dehydratase and ethanolamine ammonia-lyase, respectively. The latter analog served as an effective suicide coenzyme for both enzymes, although the partition ratio (kcat/klinact) of 630 for ethanolamine ammonialyase is much lower than that for diol dehydratase. Suicide inactivation was accompanied by the accumulation of a cob(II)amide species, indicating irreversible cleavage of the coenzyme Co-C bond during the inactivation. It was thus concluded that the bulkiness of a Co-coordinating base of the nucleotide loop is essential for both the initial activity and continuous catalytic turnovers. Since the kcat/kinact value for the imidazolyl-cobamide in diol dehydratase was 27-times higher than that for the imidazolyl trimethylene analog, it is clear that the ribosyl moiety protects the reaction intermediates from suicide inactivation. Stopped-flow measurements indicated that the rate of Co-C bond homolysis is essentially unaffected by the bulkiness of the Co-coordinating base for diol dehydratase. Thus, it seems unlikely that the Co-C bond is labilized through a ground state mechanochemical triggering mechanism in diol dehydratase. [ABSTRACT FROM AUTHOR]

The article presents a study related to major conformations of B12 which have been found in proteins. The "base-on" form in which the endogenous corrin base, dimethylbenzimidazole, is the lower axial ligand and the "base-off/His-on" conformation in which a histidine residue in the protein replaces dimethylbenzimidazole. In the latter subclass, the coordinating histidine is embedded in a conserved primary sequence motif, DXHXXG, which is absent in bacterial and human adenosyltransferases. This has led to the prediction that adenosyltransferases bind 12 in the "base-on" conformation.

The importance of each active-site residue in adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca was estimated using mutant enzymes in which one of the residues interacting with substrate and/or K+ was mutated to Ala or another amino acid residue. The Eα170A and Dα335A mutants were totally inactive, and the Hα143A mutant showed only a trace of activity, indicating that Glu-α170, Asp-α335, and His-α143 are catalytic residues. The Qα141A, Qα296A, and Sα362A mutants showed partial activity. It was suggested from kinetic parameters that Gln-α296 is important for substrate binding and Gln-α296 and Gln-α141 for preventing the enzyme from mechanism-based inactivation. The Eα221A, Eα170H, and Dα335A did not form the (αβγ)2 complex, suggesting that these mutations indirectly disrupt subunit contacts. Among other Glu-α170 and Asp-α335 mutants, EαL70D and Eα170Q were 2.2 ± 0.3% and 0.02% as active as the wild-type enzyme, respectively, whereas Dαwas totally inactive. Kinetic analysis indicated that the presence and the position of a carboxyl group in the residue α170 are essential for catalysis as well as for the continuous progress of catalytic cycles. It was suggested that the roles of Glu-α170 and Asp-α335 are to participate in the binding of substrate and intermediates and keep them appropriately oriented and to function as a base in the dehydration of the 1,1-diol intermediate. In addition, Glu-α170 seems to stabilize the transition state for the hydroxyl group migration from C2 to C1 by accepting the proton of the spectator hydroxyl group on C1. [ABSTRACT FROM AUTHOR]