The catalytic mechanism of a new orotidin$-5^{\text{'}}-$monophosphate $($OMP$)$ decarboxylase was

investigated. Based on the structure of the enzyme and the high sequence homology to OMP

decarboxylase from yeast $($Saccharomyces cerevisiae, ScOMPDC$),$ Ser $153$ and Gln $214$ were

suggested to play important roles in the catalysis. The importance of these residues was

evaluated by making the corresponding alanine variants. After a few weeks work pure

preparations of the mutants S$153$A and Q$214$A were achieved and the following experiments

were performed. The initial reaction rates were measured at different concentration of

orotidin$-5\text{'}-$monophosphate in $10mM$ MOPS, pH $7,1$ at $25C($table below$).$ The kinetic

constants $k_{\text{c}\text{a}\text{t}}$ and $K_M$ of the wild$-$type enzyme were known from earlier experiments to be $15$

$s^{-1}$ and $1,7M,$ respectively.

The reaction rates were measured in a spectrophotometer at $279nm$ by following the

disappearance of the substrate $({}_{\text{s}\text{u}\text{b}\text{s}\text{t}\text{r}\text{a}\text{t}\text{e}-}$ product $()=2400M^{-1}c{m}^{-1}).$ In a total volume of $1,0$

$ml$ the cuvette reaction mixtures contained the following volumes of the enzyme stock

solutions: $50l$ of the S$153$A variant and $15l$ of the Q$214$A variant, respectively. The

molecular weight of the wild$-$type enzyme was $64320gmo{l}^{-1}.$ The enzyme stock solutions

had the following concentrations: S$153$A $65M$; Q$214$A $0,72M.$ Cuvettes with a light path

of $10mm$ were used.

$-$Calculate $G_{ES},G_{ES}^{\text{\#}}$ and $G_{\text{k}\text{c}\text{a}\text{t}}$ and make energy$-$profile reaction$-$coordinate diagrams for

wild$-$type and mutant enzymes.