The Cys319 loop modulates the transition between dehydrogenase and hydrolase conformations in inosine 5'-monophosphate dehydrogenase.
X-ray crystal structures of enzyme-ligand complexes are widely believed to mimic states in the catalytic cycle, but this presumption has seldom been carefully scrutinized. In the case of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase (IMPDH), 10 structures of various enzyme-substrate-inhibitor complexes have been determined. The Cys319 loop is found in at least three different conformations, suggesting that its conformation changes as the catalytic cycle progresses from the dehydrogenase step to the hydrolase reaction. Alternatively, only one conformation of the Cys319 loop may be catalytically relevant while the others are off-pathway. Here we differentiate between these two hypotheses by analyzing the effects of Ala substitutions at three residues of the Cys319 loop, Arg322, Glu323, and Gln324. These mutations have minimal effects on the value of k(cat) (≤5-fold) that obscure large effects (>10-fold) on the microscopic rate constants for individual steps. These substitutions increase the equilibrium constant for the dehydrogenase step but decrease the equilibrium between open and closed conformations of a mobile flap. More dramatic effects are observed when Arg322 is substituted with Glu, which decreases the rates of hydride transfer and hydrolysis by factors of 2000 and 130, respectively. These experiments suggest that the Cys319 loop does indeed have different conformations during the dehydrogenase and hydrolase reactions as suggested by the crystal structures. Importantly, these experiments reveal that the structure of the Cys319 loop modulates the closure of the mobile flap. This conformational change converts the enzyme from a dehydrogenase into hydrolase, suggesting that the conformation of the Cys319 loop may gate the catalytic cycle.
Department of Biology, Brandeis University, Waltham, Massachusetts 02454-9110, United States.
Helen R
HR
Josephine
Kanchana R
KR
Ravichandran
Lizbeth
L
Hedstrom
D018360:Crystallography, X-Ray; D006867:Hydrolases / Q000737:chemistry / Q000378:metabolism*; D007168:IMP Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D016835:Tritrichomonas foetus / Q000201:enzymology
D010088:Oxidoreductases; D007168:IMP Dehydrogenase; D006867:Hydrolases