A role for glutamine 183 in the folate oxidative half-reaction of methylenetetrahydrofolate reductase from Escherichia coli.

The flavoprotein methylenetetrahydrofolate reductase (MTHFR) from Escherichia coli catalyzes a ping-pong reaction with NADH and 5,10-methylenetetrahydrofolate (CH2-H4folate) to produce NAD+ and 5-methyltetrahydrofolate (CH3-H4folate). This work focuses on the function of the invariant, active-site aminoacyl residue Gln183. X-ray structures of the enzyme complexes Ered(wild-type)•NADH and Eox(Glu28Gln)•CH3-H4folate indicate that Gln183 makes key hydrogen-bonding interactions with both NADH and folate in their respective half-reactions, suggesting roles in binding each substrate. We propose that the polarity of Gln183 may also aid in stabilizing the proposed 5-iminium cation intermediate during catalysis in the oxidative half-reaction with folate. We have prepared mutants Gln183Ala and Gln183Glu, which we hypothesize to have altered charge/polarity and hydrogen bonding properties. We have examined the enzymes by steady-state and stopped-flow kinetics and by measurement of the flavin redox potentials. In the reductive half-reaction, NADH binding affinity and the rate of flavin reduction have not been hindered by either mutation. By contrast, our results support a minor role for Gln183 in the oxidative half-reaction. The Gln183Ala variant exhibited a 6-10 fold lower rate of folate reduction and bound CH2-H4folate with 7-fold lower affinity, whereas the Gln183Glu mutant displayed catalytic constants within 3-fold of the wild-type enzyme.

Ultrasound-Guided Paravertebral Catheter Versus Intercostal Blocks for Postoperative Pain Control in Video-Assisted Thoracoscopic Surgery: A Prospective Randomized Trial.

OBJECTIVE: The use of continuous paravertebral (PV) catheters for management of acute postsurgical pain after video-assisted thoracoscopic surgery (VATS) has not been investigated previously as a randomized controlled trial. The purpose of this study was to compare the efficacy of an ultrasound-guided continuous PV catheter catheter infusion for postoperative pain control with single-shot intercostal blocks (ICB). DESIGN: A prospective, randomized, controlled trial. SETTING: An academic university hospital. PARTICIPANTS: Patients (≥18 years of age) who underwent a VATS procedure. INTERVENTIONS: Patients were randomized into 2 groups. Group 1 received single-shot ICB. Group 2 received an ultrasound-guided PV catheter with a continuous infusion of 0.2% ropivacaine. MEASUREMENTS AND MAIN RESULTS: There were 25 patients in group 1 and 23 patients in group 2. The maximum pain score was significantly lower in the group that received the PV catheter compared with those who received ICB during 24 to 48 hours (3.65 v 6.44, p<0.001). Seventeen patients (74%) who received PV catheters reported satisfaction with a pain control regimen compared to the 11 (44%) who received ICB (p = 0.036). In addition, during 24 to 48 hours after surgery the mean opioid use decreased significantly in the PV catheter group (14.39 v 30.50 mg morphine equivalents, p = 0.046). CONCLUSIONS: Ultrasound-guided continuous PV catheter infusions provided prolonged pain control and superior patient satisfaction compared with single-shot ICB after video-assisted thoracoscopic surgery.