posted on 2006-06-13, 00:00authored byMark A. Anderson, W. Wallace Cleland, Danny T. Huang, Camilla Chan, Maryam Shojaei, Richard I. Christopherson
In the pyrimidine biosynthetic pathway, N-carbamyl-l-aspartate (CA-asp) is converted to
l-dihydroorotate (DHO) by dihydroorotase (DHOase). The mechanism of this important reaction was
probed using primary and secondary 15N and 13C isotope effects on the ring opening of DHO using isotope
ratio mass spectrometry (IRMS). The reaction was performed at three different temperatures (25, 37, and
45 °C for hamster DHOase; 37, 50, and 60 °C for Bacillus caldolyticus), and the product CA-asp was
purified for analysis. The primary and secondary kinetic isotope effects for the ring opening of the DHO
were determined from analysis of the N and C of the carbamyl group after hydrolysis. In addition, the
β-carboxyl of the residual aspartate was liberated enzymatically by transamination to oxaloacetate with
aspartate aminotransferase and then decarboxylation with oxaloacetate decarboxylase. The 13C/12C ratio
from the released CO2 was determined by IRMS, yielding a second primary isotope effect. The primary
and secondary isotope effects for the reaction catalyzed by DHOase showed little variation between enzymes
or temperatures, the primary 13C and 15N isotope effects being approximately 1% on average, while the
secondary 13C isotope effect is negligible or very slightly normal (>1.0000). These data indicate that the
chemistry is at least partially rate-limiting while the secondary isotope effects suggest that the transition
state may have lost some bending and torsional modes leading to a slight lessening of bond stiffness at
the carbonyl carbon of the amide of CA-asp. The equilibrium isotope effects for DHO → CA-asp have
also been measured (secondary 13Keq = 1.0028 ± 0.0002, primary 13Keq = 1.0053 ± 0.0003, primary
15Keq = 1.0027 ± 0.0003). Using these equilibrium isotope effects, the kinetic isotope effects for the
physiological reaction (CA-asp → DHO) have been calculated. These values indicate that the carbon of
the amide group is more stiffly bonded in DHO while the slightly lesser, but still normal, values of the
primary kinetic isotope effect show that the chemistry remains at least partially rate-limiting for the
physiological reaction. It appears that the ring opening and closing is the slow step of the reaction.