ABSTRACT
Inosine monophosphate dehydrogenase (IMPDH) and guanosine monophosphate reductase (GMPR) belong to the same structural family, share a common set of catalytic residues and bind the same ligands. The structural and mechanistic features that determine reaction outcome in the IMPDH and GMPR family have not been identified. Here we show that the GMPR reaction uses the same intermediate E-XMP* as IMPDH, but in this reaction the intermediate reacts with ammonia instead of water. A single crystal structure of human GMPR type 2 with IMP and NADPH fortuitously captures three different states, each of which mimics a distinct step in the catalytic cycle of GMPR. The cofactor is found in two conformations: an 'in' conformation poised for hydride transfer and an 'out' conformation in which the cofactor is 6 Å from IMP. Mutagenesis along with substrate and cofactor analog experiments demonstrate that the out conformation is required for the deamination of GMP. Remarkably, the cofactor is part of the catalytic machinery that activates ammonia.
Subject(s)
GMP Reductase/metabolism , IMP Dehydrogenase/metabolism , Biocatalysis , Crystallography, X-Ray , GMP Reductase/chemistry , Guanosine Monophosphate/biosynthesis , Guanosine Monophosphate/chemistry , Guanosine Monophosphate/metabolism , Humans , IMP Dehydrogenase/chemistry , Inosine Monophosphate/chemistry , Inosine Monophosphate/metabolism , Kinetics , Models, Molecular , Molecular Structure , NADP/chemistry , NADP/metabolism , Quantum Theory , Sulfhydryl Compounds/chemistry , Sulfhydryl Compounds/metabolismABSTRACT
The reaction mechanism of cis-3-chloroacrylic acid dehalogenase (cis-CaaD) is studied using the B3LYP density functional theory method. This enzyme catalyzes the hydrolytic dehalogenation of cis-3-chloroacrylic acid to yield malonate semialdehyde and HCl. The uncatalyzed reaction is first considered, and excellent agreement is found between the calculated barrier and the measured rate constant. The enzymatic reaction is then studied with an active site model consisting of 159 atoms. The results suggest an alternative mechanism for cis-CaaD catalysis and different roles for some active site residues in this mechanism.
Subject(s)
Hydrolases/chemistry , Hydrolases/metabolism , Models, Chemical , Pseudomonas/enzymology , Catalysis , Catalytic Domain , Energy Metabolism , Hydrolysis , Pseudomonas/metabolism , Static Electricity , Substrate Specificity , Water/metabolismABSTRACT
Impaired renal development during foetal life is a proposed mechanism for adult hypertension in people born small. Whether preterm birth contributes to such adverse development is still unclear. We investigated the selective contributions from foetal growth restriction or preterm birth to renal function and volume in children with low birth weight. Three groups of 9 to 12-year-old children were studied: those born at < 32 gestational weeks (preterm, n = 39), those born at term but small for gestational age (SGA, n = 29) and those born at the term appropriate for gestational age (controls, n = 37). We estimated renal function by calculating glomerular filtration rate (GFR) and by measuring urinary proteins. Volumetric ultrasound of the kidneys was performed in 86 children (preterm, n = 33; SGA, n = 25; controls, n = 29). Estimated glomerular filtration rate (eGFR) and urinary protein patterns were similar between the groups. Kidney volume (preterm 162 ml (31); SGA 163 ml (26) and controls 182 ml (47)) was smaller in the preterm group than in the controls, but the difference was not significant when adjusted for body surface area, gender and age (P = 0.25). Total renal volume correlated to birth weight (r = 0.23, P = 0.03). No significant differences were found in renal function or volume between the three groups at school age.
Subject(s)
Fetal Growth Retardation/epidemiology , Fetal Growth Retardation/pathology , Infant, Premature , Infant, Small for Gestational Age , Kidney/abnormalities , Blood Pressure , Child , Female , Fetal Growth Retardation/physiopathology , Glomerular Filtration Rate , Humans , Infant, Newborn , Kidney/physiology , Kidney Function Tests , Male , Risk FactorsABSTRACT
The reaction mechanism of 4-oxalocrotonate tautomerase (4-OT) is studied using the density functional theory method B3LYP. This enzyme catalyzes the isomerisation of unconjugated alpha-keto acids to their conjugated isomers. Two different quantum chemical models of the active site are devised and the potential energy curves for the reaction are computed. The calculations support the proposed reaction mechanism in which Pro-1 acts as a base to shuttle a proton from the C3 to the C5 position of the substrate. The first step (proton transfer from C3 to proline) is shown to be the rate-limiting step. The energy of the charge-separated intermediate (protonated proline-deprotonated substrate) is calculated to be quite low, in accordance with measured pKa values. The results of the two models are used to evaluate the methodology employed in modeling enzyme active sites using quantum chemical cluster models.
