thymidylate synthase ( tsase ) catalyzes 
 the intracellular de novo formation of thymidylate 
 ( a dna building block ) 
 in most living organisms , making it a common target for chemotherapeutic 
 and antibiotic drugs . 
 two mechanisms have been proposed for the rate - limiting 
 hydride transfer step in tsase catalysis : a stepwise mechanism in 
 which the hydride transfer precedes the cleavage of the covalent bond 
 between the enzymatic cysteine and the product and a mechanism where 
 both happen concertedly . striking similarities between the enzyme - bound 
 enolate intermediates formed in the initial and final step of the 
 reaction supported the first mechanism , while qm / mm calculations favored 
 the concerted mechanism . here , we experimentally test these two possibilities 
 using secondary kinetic isotope effect ( kie ) , mutagenesis study , and 
 primary kies . 
 the findings support the concerted mechanism and demonstrate 
 the critical role of an active site arginine in substrate binding , 
 activation of enzymatic nucleophile , and the hydride transfer studied 
 here . 
 the elucidation of this reduction / substitution sheds light on 
 the critical catalytic step in tsase and may aid future drug or biomimetic 
 catalyst design .