posted on 2019-11-06, 18:37authored byTsuyoshi Egawa, Hua Deng, Eric Chang, Robert Callender
We
investigate how isotopic labeling of the enzyme lactate dehydrogenase
(LDH) affects its function. LDH is of special interest because there
exists a line of residues spanning the protein that are involved in
the transition state (TS) of the chemical reaction coordinate (so-called
promoting vibration). Hence, studies have been carried out on this
protein (as well as others) using labeled protein (so-called heavy
protein) along with measurements of single turnover kcat yielding a KIE (=kcatlight/kcatheavy) aimed
at understanding the effect of labeling generally and more specifically
this line of residues. Here, it is shown that 13C, 15N, and 2H atom labeling of hhLDH (human heart)
affects its internal structure which in turn affects its dynamics
and catalytic mechanism. Spectral studies employing advanced FTIR
difference spectroscopy show that the height of the electronic potential
surface of the TS is lowered (probably by ground state destabilization)
by labeling. Moreover, laser-induced T-jump relaxation kinetic spectroscopy
shows that the microsecond to millisecond nuclear motions internal
to the protein are affected by labeling. While the effects are small,
they are sufficient to contribute to the observed KIE values as well
or even more than promoting vibration effects.