Biochemistry: The Chemical Reactions of Living Cells, Volume 2 - D. Metzler 1980

Types of Enzyme-Catalyzed Reactions
Addition and Elimination Reactions
Addition to double bonds adjacent to carboxyl groups

Biochemical reactions frequently involve addition to C=C bonds conjugated not with a true carbonyl group, but with a —COO^- group, which is a considerably weaker electron acceptor. When this group is part of a protein molecule, it may exist in a protonated state, making it a more effective electron acceptor. Nevertheless, the question arises as to whether such Enzymes operate via the mechanism shown in equation (7-41). Indeed, various experimental data compel us to assume a completely distinct mechanism—specifically, the one postulated for the conventional non-enzymatic Hydration of alkenes. An example of such a reaction is the hydration of Ethylene with hot Water in the presence of dilute sulfuric acid as a catalyst, which forms The basis of the industrial method for ethanol production:

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A proton adds to the $\pi$-electrons of the double bond, and the resulting carbonium ion readily abstracts a hydroxyl ion from water. At the same time, the direct Addition of an OH^- group to form a carbonium ion is hindered because the ethylene molecule lacks a carbonyl group adjacent to the double bond capable of stabilizing the negative charge.

a. Fumarase [111]

Among enzymes of this group, fumarase (fumarate hydratase, Chapter 6, Section D,2 and E,5) is perhaps the best studied. It is a tetramer with a Molecular Weight of ~ 200,000 and is characterized by a turnover number of about 2∙10³ s^-1. The product of the reaction catalyzed by fumarase is L-malate (R-malate). If the reaction is carried out in ²H₂O, the ²H atom is incorporated into the pro-R position; that is, the proton adds to the re-face of the trigonal carbon atom:

To yield L-malate, the hydroxyl group must add to the opposite side of the double bond. In both enzymatic and non-enzymatic reactions, such anti (trans) addition is far more prevalent than The addition of H and OH (or —Y) to the same face (syn or cis addition, addition to the same side of the double bond). With the exception of two reactions that yield unusual structures, all enzymatic Addition and elimination reactions studied thus far involve anti-addition, where the proton approaches from the re-face. This has led to the suggestion that a single, fundamentally conserved mechanism has been evolutionarily fixed [112].

The pH dependence profile of fumarase-catalyzed reactions indicates the involvement of both acidic and basic groups at the Active Site (Chapter 6, Section E,5). However, these data do not yet provide conclusive evidence to distinguish between an anionic mechanism and a carbonium ion mechanism. The assumption that C—H bond Cleavage is not the rate-limiting step is based on the following observation: malate containing ²H in the pro-R position is dehydrated at the same rate as ordinary malate. If an anionic mechanism [equation (7-41)] were operative, this could imply that the ²H from the pro-R position of the selectively labeled malate is removed in a fast step and that the departure of the OH^- group proceeds much more slowly. If so, ²H should "wash out" from L-malate faster than would occur if the reaction proceeded via conversion to fumarate followed by rehydration to malate. In reality, the opposite is observed: the hydroxyl group is eliminated rapidly, whereas ²H is released more slowly. These results point to a mechanism involving a carbonium ion:

Although there is no primary kinetic isotope effect when measuring reaction rates, distinct secondary isotope effects have been observed when replacing the hydrogen atom at C-2 or the pro-S hydrogen atom at C-3 of malate with ²H or ³H. For instance, the k(¹Н)/k(²Н) ratio is 1.09 for hydrogen atoms located at both the pro-S position and the C-3 position [113]. These findings appear to support, first, the operation of a carbonium ion mechanism, and second, the fact that step b in equation (7-45) is rate-limiting (see also Section C, 4,6). A comparison of the V_max values for the hydration of fumarate, fluorofumarate, and difluorofumarate (104, 410, and 86 µmol ∙ min^-1 ∙ mg^-1, respectively) also likely indicates a carbonium ion mechanism (students are encouraged to attempt to interpret these data before consulting the original paper) [114].

It has recently become apparent that protons capable of transferring from the substrate to basic protein groups do not necessarily exchange rapidly with the solvent (see Section I,2). Indeed, it has been demonstrated that the proton abstracted by fumarase from malate is retained by the enzyme for a relatively long period of time. The rate of proton exchange between malate and the solvent is lower than the rate of exchange between a bound fumarate ion on the enzyme surface and another substrate molecule present in the medium [115]. Thus, it turns out that the overall exchange rate is determined by the rate of product release from the enzyme molecule, and we still do not know whether proton removal precedes or follows OH^- elimination. However, a third possibility exists—the proton and the hydroxyl group may add simultaneously via a concerted mechanism [115].

b. Enolase

The dehydration of 2-phosphoglycerate to yield phosphoenolpyruvate, a phosphorylated derivative of the enol form of pyruvic acid, is catalyzed by the enzyme enolase and represents a key reaction in Carbohydrate METABOLISM:

Based on isotope exchange data obtained for this enzyme, it has been proposed that a proton is rapidly removed to form a carbanion intermediate, The breakdown of which is the rate-limiting step [116]. This enzyme Functions only when complexed with a metal ion [117], typically Mg²⁺ or Mn²⁺. NMR water proton relaxation studies indicate that the Mn²⁺ ion coordinates with two rapidly exchanging water molecules in the free enzyme [118]. Upon substrate binding, one of these water molecules may be immobilized and participate in the addition reaction. The phosphate group of the substrate can act as a general base catalyst, facilitating proton abstraction from a water molecule [118]:

c. Aconitase

Another enzyme in this group, aconitase (aconitate hydratase), catalyzes two reactions: (a) the dehydration of citrate to yield cis-aconitate, and (b) a rehydration proceeding via a different mechanism to yield isocitrate:

Both reactions are completely stereospecific. In the first reaction (step a), the pro-R proton is removed from C-4 (Stereochemical Numbering) of citrate, whereas step b yields threo-D_s-isocitrate. Proton addition occurs from the re-face in both cases.

Much like fumarase, the enzyme retains the abstracted proton for a considerable length of time (up to 7–10^-1 s), which occasionally allows the cis-aconitate molecule to diffuse away from the enzyme surface and be replaced by another (if cis-aconitate is present in excess). As a result, a new cis-aconitate molecule sometimes acquires a proton (intermolecular proton transfer). The proton abstracted from citrate frequently returns to the molecule [step b in equation (7-47)], but as indicated by the dashed arrow leading to the species depicted in the center of equation (7-47), the position where the returning proton attaches differs from THE POSITION OF its abstraction. Evidently, following the initial proton abstraction, the resulting cis-aconitate "tumbles" into an orientation that permits rehydration involving the same groups that participated in dehydration, but leading to The formation of a new product [119]. It is quite possible that this complex mechanism accounts for the low turnover number (15 s^-1).

The aconitase molecule contains a ferrous ion (Fe²⁺) that is essential for catalytic activity. Although it has been suggested that the iron ion may participate in a redox process (operating via a mechanism distinct from the one discussed above), it is more likely that the iron ion facilitates substrate binding to the enzyme and the generation of a hydroxyl ion, analogously to what has been postulated for the Zn²⁺ ion in Carbonic anhydrase (Section 3.2) [120].

d. Addition and elimination of other nucleophiles

Besides hydroxyl groups, other groups can also add to (or be eliminated from) positions $\beta$ to a carboxyl group. For instance, the bacterial enzyme aspartase catalyzes the addition of ammonia to fumarate to yield L-aspartate—a reaction analogous to that catalyzed by fumarase. This process exhibits trans-addition, and kinetic isotope effects point to a mechanism involving a carbonium ion [121]. Meanwhile, $\beta$-methylaspartase, which catalyzes a similar addition, promotes rapid Deuterium Exchange between water and the substrate protons [122].

1,4-linked uronic acid polymers, such as hyaluronic acid, dermatan sulfate (Fig. 2-16), and Pectins (Chap. 2, Sec. B,3), can be cleaved by a group of bacterial enzymes that utilize an elimination mechanism [123]. The geometry of the galacturonic acid units joined by ß-linkages in the pectin molecule is favorable for the trans-elimination of 5-H and the O-glycosidic group at position 4:

However, the corresponding hyaluronidase acting on glucuronic acid residues promotes cis-elimination. Based on these results, it can be suggested that the activating effect of the carboxyl group, which facilitates the abstraction of the 5-hydrogen atom as a proton, is of primary importance [124], and that an anionic intermediate is formed during this reaction. In this case, elimination of the ß-substituent can proceed from either an equatorial or an axial position.

Addition reactions to the double bond catalyzed by certain cis-trans isomerases have an interesting application [125]. These enzymes contain sulfhydryl groups. One isomerase converts maleate into fumarate with a turnover number of 300 s^-1; related enzymes catalyze the isomerization of maleylacetate and maleylpyruvate into the corresponding fumaryl derivatives:

According to current models, the —SH groups of the enzyme add to the double bond. Subsequently, rotation can occur in the enolic intermediate. Thiocyanate ion catalyzes the isomerization of maleic acid in the absence of enzymes via a mechanism analogous to the enzymatic one.