Tuning of the H-transfer coordinate in primitive versus well-evolved enzymes.

MATERIALS AND METHODS Materials Reagent-grade chemicals were used as received unless specified otherwise. βNicotinamide [U-C]adenine dinucleotide (50 μCi) was purchased from Amersham Pharmacia (specific radioactivity >220 mCi/mmol), and Congo Red was obtained from Aldrich. Alcohol dehydrogenase from Thermoanaerobium brockii (tbADH), and 2propanold8 were purchased from Sigma. 7,8-Dihydrofolate (DHF) was prepared by dithionite reduction of folic acid as described by Blakely.