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Accounts of Chemical Research y-phosphoryl group remains coordinated by the enzyme-bound Mn2'. The distance to the reaction-center a-phosphorus atom (4.2 A) is most simply explained by the rapid averaging of 515% inner-sphere coordination with 285 % second-sphere coordination. The resulting polyphosphate conformation is puckered and somewhat strained. Hence an important role of the divalent cation activator in catalysis is to assist the departure of the leaving pyrophosphate group by y coordination, and possibly to facilitate nucleophilic attack on the aphosphorus atom by strain and by hydrogen bonding through a coordinated water ligand."A second difference between the binary and ternary complexes is in the conformational angle x about the thymine-deoxyribose bond of dTTP. The x value of 40 f 5' in the binary complex increases to 90 f 5' in the ternary complex ( Figure 7A). Similarly, a 90° torsion angle is also found for the purine nucleotide substrate Mn-dATP when bound to DNA polymerase ( Figure 7B)." Interestingly, the latter torsion angle of 90' is that found for the deoxynucleotidyl units in double-helical DNA. Hence the binding of the substrate Mn-dTTP to the enzyme, DNA polymerase, in the absence of template, has changed the substrate conformation to that of a nucleotidyl unit in the product-double-helical DNA.When the structure of enzyme-bound Mn-dTTP is superimposed by computer onto the double-helical structure of DNA-B ( Figure 7C), the resulting location of the a-phosphorus atom and the leaving pyrophosphate group of the bound substrate relative to the attacking 3'-OH group of the preceding nucleotide unit is consistent only with an in-line nucleophilic displacement on the cr phosphorus.'' Hence the biosynthesis of nucleic acids, like their h y d r~l y s i s ,~~ appears to proceed by an in-line mechanism.The selection by the enzyme of those substrate conformations that fit into the double helix would amplify the Watson-Crick base-pairing scheme and would explain the low error rates of DNA ~olymerases~~ which are at least two orders of ma itude below those of base-pairing alone.46 ConclusionsNumerous examples have been provided which establish that the average conformation of a flexible substrate, when bound to an enzyme, generally differs from that of the free substrate in solution (Figures 1, 4, and 7).Second-sphere enzyme-metal-(H20)-substrate complexes are used by enzymes to polarize carbonyl groups (six examples, Figures 1 and 2) and to position phosphoryl groups for nucleophilic attack (four examples, Figures 4-7). In the case of pyruvate kinase, an additional metal interacts directly with the ATP (Figure 5 ) .On two-substrate enzymes, such as dehydrogenases (Figure l), kinases (Figure 5 ) , and even on a biotin enzyme, (Figure 3), close proximity of the two bound substrates is observed, in some cases approaching molecular contact.The inner coordination sphere of a metal is used to facilitate the departure of the leaving group in DNA polymerase (Figure 7).

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The threo-anti-Markownikoff and threo-Markownikoff adducts are the products of addition of 4-chloroand 2,4-dinitrobenzenesulfenyl chloride to cis-I-phenylpropene while only the erythro Markownikoff adduct is the product of addition to trans-I-phenylpropene. The addition reaction follows second order kinetics, first order in both olefin and arylsulfenyl chloride, with the rate of addition to the trans-oleiin faster than to the cis. These results are consistent with a mechanism involving bridged transition states in both the rate and product determining steps.Les produits d'addition fhrio-anti-Markownikoff et fhrko-Markownikoff sont obtenus par I'action du chlorure de chloro-4 et de dinitro-2,4 benzene sulfenyle sur le phknyl-1 propene cis tandis que la meme reaction sur le compose trans conduit uniquement au compose d'addition erythro-Markownikoff. La reaction d'addition suit des cinetiques d'ordre deux; I'ordre etant de un a la fois pour I'olefine et pour le chlorure d'arylsulfenyle, la vitesse de I'addition sur I'olefine trans est plus rapide que pour le cis. Ces resultats sont compatibles avec un mecanisme impliquant des etats de transition pontes a la fois pour les etapes determinantes de la cinetique et du produit.

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