We used a multiplexed approach based on flow-stretched DNA to monitor the enzymatic digestion of lambda-phage DNA by individual bacteriophage lambda exonuclease molecules. Statistical analyses of multiple single-molecule trajectories observed simultaneously reveal that the catalytic rate is dependent on the local base content of the substrate DNA. By relating single-molecule kinetics to the free energies of hydrogen bonding and base stacking, we establish that the melting of a base from the DNA is the rate-limiting step in the catalytic cycle. The catalytic rate also exhibits large fluctuations independent of the sequence, which we attribute to conformational changes of the enzyme-DNA complex.

We show that the mechanisms of DNA-dependent and -independent dTTP hydrolysis by the gene 4 protein of bacteriophage T7 differ in the pathways by which these reactions are catalyzed. In the presence of dTTP, gene 4 protein monomers assemble as a ring that binds single-stranded DNA and couples the hydrolysis of dTTP to unidirectional translocation and the unwinding of duplex DNA. When mixing wild-type monomers with monomers lacking the catalytic base for the dTTPase reaction, we observe that each wild-type subunit hydrolyzes dTTP independently in the absence of single-stranded DNA. Conversely, when either these catalytically inactive monomers or altered monomers incapable of binding single-stranded DNA are mixed with wild-type monomers, a small fraction of altered to wild-type monomers causes a sharp decline in DNA-dependent dTTP hydrolysis. We propose that sequential hydrolysis of dTTP is coupled to the transfer of single-stranded DNA from subunit to adjacent subunit.

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The DNA helicase encoded by gene 4 of bacteriophage T7 couples DNA unwinding to the hydrolysis of dTTP. The loss of coupling in the presence of orthovanadate (Vi) suggests that the ␥-phosphate of dTTP plays an important role in this mechanism. The crystal structure of the hexameric helicase shows Arg-522, located at the subunit interface, positioned to interact with the ␥-phosphate of bound nucleoside 5 triphosphate. In this respect, it is analogous to arginine fingers found in other nucleotide-hydrolyzing enzymes. When Arg-522 is replaced with alanine (gp4-R522A) or lysine (gp4-R522K), the rate of dTTP hydrolysis is significantly decreased. dTTPase activity of the altered proteins is not inhibited by Vi, suggesting the loss of an interaction between Vi and gene 4 protein. gp4-R522A cannot unwind DNA, whereas gp4-R522K does so, proportionate to its dTTPase activity. However, gp4-R522K cannot stimulate T7 polymerase activity on double-stranded DNA. These findings support the involvement of the Arg-522 residue in the energy coupling mechanism.

Metal ligands of the VO(2+)-adenosine diphosphate (ADP) complex bound to high-affinity catalytic site 1 of chloroplast F(1) adenosine triphosphatase (CF(1) ATPase) were characterized by electron paramagnetic resonance (EPR) spectroscopy. This EPR spectrum contains two EPR species designated E and F not observed when VO(2+)-nucleotide is bound to site 3 of CF(1). Site-directed mutations betaE197C, betaE197D, and betaE197S in Chlamydomonas CF(1) impair ATP synthase and ATPase activity catalyzed by CF(1)F(o) and soluble CF(1), respectively, indicating that this residue is important for enzyme function. These mutations caused large changes in the (51)V hyperfine tensors of VO(2+)-nucleotide bound to site 1 but not to site 3. Mutations to the Walker homology B aspartate betaD262C, betaD262H, and betaD262T of Chlamydomonas CF(1) caused similar effects on the EPR spectrum of VO(2+)-ADP bound to site 1. These results indicate that the conversion of the low-affinity site 3 conformation to high-affinity site 1 involves the incorporation betaE197 and betaD262 as metal ligands.