Sub-ångström-resolution crystallography reveals physical distortions that enhance reactivity of a covalent enzymatic intermediate

Abstract

It is recognized widely that enzymes promote reactions by providing a pathway that proceeds through a transition state of lower energy. In principle, further rate enhancements could be achieved if intermediates are prevented from relaxing to their lowest energy state, and thereby reduce the barrier to the subsequent transition state. Here, we report sub-ångström-resolution crystal structures of genuine covalent reaction intermediates of transketolase. These structures reveal a pronounced out-of-plane distortion of over 20° for the covalent bond that links cofactor and substrate, and a specific elongation of the scissile substrate carbon-carbon bond (d > 1.6 Å). To achieve these distortions, the protein's conformation appears to prevent relaxation of a substrate-cofactor intermediate. The results implicate a reduced barrier to the subsequent step that is consistent with an intermediate of raised energy and leads to a more efficient overall process.

Structural Views along the Mycobacterium tuberculosis MenD Reaction Pathway Illuminate Key Aspects of Thiamin Diphosphate-Dependent Enzyme Mechanisms

“…The thiazolium ring is slightly distorted such that C2a, expected to lie in the thiazolium ring plane, is about 0.3 Å out of plane, displaced in a direction away from the AP N4 0 atom. Similar out-of-plane displacement has been seen in high-resolution structures of the equivalent intermediates of other ThDP-dependent enzymes (Berthold et al, 2007;Ludtke et al, 2013). A crucial feature in the Mtb-MenD structure, however, is that the C2a-OH is tilted further away from the AP N4 0 to a distance (4.5 Å ) too great for attack, thus preventing premature product release, as described later.…”

Progress in the experimental observation of thiamin diphosphate-bound intermediates on enzymes and mechanistic information derived from these observations

“…We note that with this substitution, there are several examples now in the literature indicating that the C2-C2a bond may be out of the plane of the thiazolium ring [71,72,87]. This certainly suggests, although does not prove, that there is van der Waals repulsion between the C2a substituent and the 4 0 -imino nitrogen under these conditions.…”

Akt Phosphorylation and Regulation of Transketolase Is a Nodal Point for Amino Acid Control of Purine Synthesis

“…We propose that Thr 382 phosphorylation changes the conformation of the Pyr domain, thereby altering the dynamic state of the Pyr-PP dimer interface and increasing binding affinities for TPP and substrates. Supporting this hypothesis are recent sub-angstrom resolution crystallographic studies that show TKT activity to be particularly sensitive to conformational changes in the cofactor and substrate binding channels (Ludtke et al, 2013). …”