Enzyme-proton complexes

Fig. 6.58 Enzyme-proton complexes. (Adapted from http //bmbiris.bmb.uga.edu/wampler/8010/ QandA3.gif).

FIGURE 5.8. A downhill trajectory for the proton transfer step in the catalytic reaction of trypsin. The trajectory moves on the actual ground state potential, from the top of the barrier to the relaxed enzyme-substrate complex. 1, 2, and 3 designate different points along the trajectory, whose respective configurations are depicted in the upper part of the figure. The time reversal of this trajectory corresponds to a very rare fluctuation that leads to a proton transfer from Ser 195 to His 57. 

A case similar to the slow, practically irreversible inhibition of jack bean a-D-mannosidase by swainsonine is represented by the interaction of castanospermine with isomaltase and rat-intestinal sucrase. Whereas the association constants for the formation of the enzyme-inhibitor complex were similar to those of other slow-binding glycosidase inhibitors (6.5 10 and 0.3 10 M s for sucrase and isomaltase, respectively), the dissociation constant of the enzyme-inhibitor complex was extremely low (3.6 10 s for sucrase) or could not be measured at all (isomaltase), resulting in a virtually irreversible inhibition. Danzin and Ehrhard discussed the strong binding of castanospermine in terms of the similarity of the protonated inhibitor to a D-glucosyl oxocarbenium ion transition-state, but were unable to give an explanation for the extremely slow dissociation of the enzyme-inhibitor complex. 

Structures of actual enzyme-substrate complexes are generally difficult to determine, because the reaction occurs too quickly, but techniques now available occasionally enable study of these complexes [53]. Protein X-ray crystallography has several limitations, for example, it often gives little or no information about the positions of protons (because of the low electron density of hydrogen atoms) in a particular protein. This can cause prob-... 

Thymidylate synthase also catalyzes an exchange of tritium of [5- H]dUMP for water protons in the absence of CH2H4folate. The turnover number for this exchange reaction is about 1/45,000 that of dTMP formation, and the Kjn is 1.2 X 10 M, about the same as the of the enzyme-dUMP complex estimated by equilibrium dialysis. The exchange reaction provided compelling evidence that the enzymic reaction involves attack of an enzyme nucleophile on the 6 position of dUMP to pro-... 

According to Supuran and co-workers (199,204), an enzyme-activator complex may be formed in which the activator facilitates the proton transfer. A general reaction scheme is presented in Scheme 9. 

Other mechanisms The active site can provide catalytic groups that enhance the probability that the transition state is formed. In some enzymes, these groups can participate in general acid-base catalysis in which amino acid residues provide or accept protons. In other enzymes, catalysis may involve the transient formation of a covalent enzyme-substrate complex. 

Often only one of the ionic forms of Eq. 3-3 will be important in a biochemical reaction. A particular ionic species may be the substrate for an enzyme. Likewise, an enzyme-substrate complex in only a certain state of protonation may react to given products. In these cases, and whenever pH affects an equilibrium, it is useful to relate the concentration [A ] of a given ionic form of a compound to the total of all ionic forms [A]t using Eqs. 3-4 and 3-5. 

The active site contains two Zn2+ ions and one Mg2+ ion which are held by imidazole and carboxylate groups. The inorganic phosphate in an enzyme-product complex is bound to both zinc ions (Fig. 12-23). The Ser 102 side chain is above one Zn. In the enzyme-P intermediate it would be linked to the phospho group as an ester which would then be hydrolyzed, reversibly, by a water molecule bound to Zn.712 713a This water presumably dissociates to Zn+-OH and its bound hydroxyl ion carries out the displacement. This reaction may be preceded by a proton transfer to an oxygen atom of the phospho group.714... 

The enzyme is a hexamer, actually a dimer of trimers made up of 291-residue polypeptide chains.28 Aceto-acetyl-CoA is a competitive inhibitor which binds into the active site and locates it. From the X-ray structure of the enzyme-inhibitor complex it can be deduced that the carboxylate group of E144 abstracts a proton from a water molecule to provide the hydroxyl ion that binds to the P position (Eq. 13-6, step a) and that the E164 carboxyl group donates a proton to the intermediate enolate anion in step b.28 The hydroxyl group... 

In order to show that the origin of this difference is not a function of the particular substrate analogue used, similar NMR relaxation studies have been performed with dimethyl sulfoxide (DMSO)1401 since the crystal structure of the enzyme-NADH-DMSO ternary complex is well resolved.1366 From the relaxation data, the distance between the methyl protons of DMSO and Co11 was calculated to be 8.9 0.9 A, again too great for direct coordination of the sulfoxide group to the metal ion. Since the cobalt enzyme appears to be functionally similar to the native enzyme, the difference is unlikely to be a direct result of substitution. One possibility is that there may actually be a difference between the solution and crystalline structure of the enzyme ternary complex, particularly since it is well established that the crystalline enzyme is 1000 times less active than in solution.1402... 

The catalytically important His-195 is unusually reactive toward diethylpyro-carbonate. This enabled the pKa in both the apo and holoenzymes to be determined directly from the pH dependence of the rate of modification (the pKa — 6.7).58 There is evidence that lactate binds preferentially to the holoenzyme containing the un-ionized histidine, whereas pyruvate binds preferentially to the enzyme-NADH complex containing protonated histidine. 

The activities of many enzymes vary with pH in the same way that simple acids and bases ionize. This is not surprising, since, as we saw in Chapter 1, the active sites generally contain important acidic or basic groups (Table 5.1). It is to be expected that if only one protonic form of the acid or base is catalyti-cally active, the catalysis will somehow depend on the concentration of the active form. In this chapter we shall see that kcM, KM, and kcJKM are affected in different ways by the ionizations of the enzyme and enzyme-substrate complex. 

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