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abstracts
Chitinases are enzymes that hydrolyze chitin, a glucosamine polymer synthesized by lower organisms for structural purposes [1]. While humans do not synthetize chitin, they express two active chitinases, Chitotriosidase (hCHIT1) and Acidic Mammalian Chitinase (hAMCase). Both enzymes attracted attention due to their upregulation in immune system disorders [2,3]. They consist of a catalytic domain of 39 kDa and a chitin binding domain, joined by a hinge. The active site shows a cluster of three conserved acidic residues, E140, D138 and D136, linked by H-bonds, where D138 and E140 are involved in the hydrolysis reaction [1,3]. To increase our knowledge on the catalytic mechanism of human chitinases, we conducted a detailed structural analysis on hCHIT1. For this, we have improved the X-ray resolution of the apo hCHIT1 catalytic domain to 1Å. We investigated the protonation state on the catalytic site and detected a double conformation of D138, one in contact with D136 and a second one in contact with E140. Our analysis revealed for the first time different protonation states for each conformation of D138 (fig1). Interestingly, our X-ray data suggest that the catalytic E140, supposed to donate a proton in the catalytic reaction, is deprotonated in the apo form. To gain insight on the proton transition pathway during the hydrolysis, we have solved the X-ray structure of hCHIT1 complexed with the substrate at 1.05 Å. In comparison with the apo form, this structure shows a rearrangement of the protonation states of the catalytic triad triggered by the binding of the substrate. Our results led us to suggest a new hydrolysis model involving changes in the hydrogen bond network of the catalytic triad accompanied by a flip of D138 towards D136. This contributes to protonate E140, which then donates the proton to the substrate. To confirm the role of the active site's hydrogen network, we are currently studying CHIT1 by neutron crystallography and quantum mechanics.
abstracts
Early neutron crystallography studies replaced hydrogen with deuterium by soaking the crystal in heavy water prior to data collection, which exchanged labile hydrogen atoms (OH, NH, and SH) and solvent molecules only. Carbon bonded hydrogen atoms were not replaced, and their negative scattering density resulted in cancellation in nuclear density maps with resolution worse than 1.8 Å. Furthermore complications arise due to partial exchange, where deuterium is present in some unit cells and hydrogen in others. More recently it has become possible to completely replace hydrogen with deuterium through expression in a deuterated medium, using facilities such as the Deuteration Laboratory (DLAB) in Grenoble. As this is a complex and expensive task, the question arises as to the importance of its use. As well as allowing the study of radically smaller crystals (<0.05mm3), it also has the possibility to avoid the cancellation problems discussed above. We have obtained data from high quality crystals of partially hydrogenated type III antifreeze protein, where methyl protonated valine and leucine residues were incorporated into the perdeuterated protein. This provides an excellent opportunity to assess the effects of negative scattering from hydrogen atoms not only on the visibility of neighbouring carbon atoms but also on water molecules in close vicinity. The observation of these cancellation effects gives a further reason to use full deuteration in neutron protein crystallography.
