Histidine-α143 Assists 1,2-Hydroxyl Group Migration and Protects Radical Intermediates in Coenzyme B₁₂-Dependent Diol Dehydratase

Abstract

Diol dehydratase of Klebsiella oxytoca contains an essential histidine residue. Its X-ray structure revealed that the migrating hydroxyl group on C2 of substrate is hydrogen-bonded to Hisalpha143. Mutant enzymes in which Hisalpha143 was mutated to another amino acid residue were expressed in Escherichia coli, purified, and examined for enzymatic activity. The Halpha143Q mutant was 34% as active as the wild-type enzyme. Halpha143A and Halpha143L showed only a trace of activity. Kinetic analyses indicated that the hydrogen bonding interaction between the hydroxyl group on C2 of substrate and the side chain of residue alpha143 is important not only for catalysis but also for protecting radical intermediates. Halpha143E and Halpha143K that did not exist as (alphabetagamma) 2 complexes were inactive. The deuterium kinetic isotope effect on the overall reaction suggested that a hydrogen abstraction step is fully rate-determining for the wild type and Halpha143Q and partially rate-determining for Halpha143A. The preference for substrate enantiomers was reversed by the Halpha143Q mutation in both substrate binding and catalysis. Upon the inactivation of the Halpha143A holoenzyme by 1,2-propanediol, cob(II)alamin without an organic radical coupling partner accumulated, 5'-deoxyadenosine was quantitatively formed from the coenzyme adenosyl group, and the apoenzyme itself was not damaged. This inactivation was thus concluded to be a mechanism-based inactivation. The holoenzyme of Halpha143Q underwent irreversible inactivation by O 2 in the absence of substrate at a much lower rate than the wild type.

But‐3‐ene‐1,2‐diol: A Mechanism‐Based Active Site Inhibitor for Coenzyme B12‐Dependent Glycerol Dehydratase

“…[4] The mechanism of action of coenzyme B 12 -dependent diol/ glycerol dehydratase has been elucidated by stereochemical studies, [5,6] experimental [7] and theoretical models, [8] and holoenzyme crystal structures. [9] The diol (for example, propane-1,2-diol), bound at the active site by a potassium ion and selected protein residues, is subject to attack at a C-1 hydrogen atom by the 5'-deoxyadenosyl radical released from the co-A C H T U N G T R E N N U N G enzyme by homolysis of the CoÀC s-bond. The resulting substrate radical undergoes a 1,2-oxygen shift [10] enabled by the "push-pull" effect of weakly acidic and basic groups of the enzyme.…”

Roles of adenine anchoring and ion pairing at the coenzyme B12‐binding site in diol dehydratase catalysis

“…The adenosyl group‐derived product(s) formed from AdoCbl in the inactivation of a mutant holoenzyme during catalysis was identified as described previously [46]. Substrate‐free apoenzyme (1.0 mg, 4.6 nmol) was incubated at 37 °C for 30 min in the dark with 15 μ m AdoCbl in the presence of 0.1 m 1,2‐propanediol.…”

“…The enzyme purified as described above was not appropriate for the resolution experiment, because Brij35 was salted out in the process of acid ammonium sulfate fractionation and was no longer effective for solubilizing the enzyme. Instead, trypsin‐solubilized Sα224A apoenzyme was prepared as described previously [46] and successfully used in the resolution experiments. The Sα224A holoenzyme inactivated during catalysis was obtained by the incubation of trypsin‐solubilized apoenzyme (1.5 mg) at 37 °C for 30 min with AdoCbl with the substrate 1,2‐propanediol, followed by dialysis, and the inactivated holoenzyme obtained was then resolved by acid ammonium sulfate treatment, as described previously [47].…”

Glycerol as a Substrate and Inactivator of Coenzyme B12‐Dependent Diol Dehydratase

“…C1), and that the barrier for this process is relatively low. [55,59] If hydrogen abstraction from GOL occurs from C3, however, the expected higher energy barrier for OH migration (C3!C2) could result in the reactant radical being trapped in a deep potential energy well (i. e. high forward and reverse barriers). The same expectation is not reasonable for PDO as a substrate.…”

“…As noted, Glu170, His143 and Ca 2 + are responsible for stabilizing the TS2 C1 transition state. [19,[55][56][57][58][59]63] Because of its medial proximity in the active site and its directionality towards the C2À OH group, His143 likely stabilizes TS2 C3 (as it does in TS2 C1 ) through the so-called "push" effect. The role of stimulating the "pull" effect, which is achieved by Glu170 in TS2 C1 , is likely assumed by Asp335 along the C3 path, as both residues have carboxylic moieties interacting with a spectator OH group (at C1 or C3, depending on the pathway).…”