In-line mechanism

One of the questions that is commonly addressed in mechanistic proposals is how is the active site water activated for nucleophilic attack on the phosphodi-ester bond Numerous combinations of amino acid side chains and zinc ions have been proposed for this role, but there has been little consensus. Critical to all the general base hypotheses is a quite reasonable assumption about catalysis by PLC5c The nucleophilic attack on the phosphodiester moiety proceeds via an in-line mechanism resulting in stereochemical inversion of configuration at phosphorus [86]. While this assumption is consistent with the position of the active site water molecules in the PLCBc-phosphonate inhibitor complex [45], it has not yet been established experimentally. This structure provides a detailed picture of how the amino acid side chains of Glul46, Glu4, Asp55, and the zinc ions interact with the phosphonate inhibitor (Fig. 12), so mechanistic hypotheses now have a structural basis. 

Is the favored in-line mechanism suggested by the geometry of the enzyme and the substrate in both stages of the reaction ... 

Fig. 19. The in-line mechanism propiosed for RNase A by Mathias and Rabin (Findlay etd 1962) as modified by Roberts et al. (1969). Only the first stage, formation of the cyclic intermediate, is shown. The second stage is the reverse of the first, but with R = H. The residues implicated in catalysis by this study are shown. This is the mechanistic proposal most consistent with the structural data summarized in this article.

IN-LINE MECHANISM STEREOCHEMISTRY WALDEN INVERSION INVERTASE SUCRASE... 

Recently the related cyclization of the phenyl ester of c/j-tetrahydrofuran-3,4-diol monophosphate to the corresponding five-membered phosphate with loss of phenol has been shown to be subject to general catalysis by imidazole132. This reaction serves as a model for the first step in the action of ribonuclease which leads to the formation of the nucleoside 2 ,3 -cyclic phosphate. The actual details of the transition state leading to the cyclic phosphate as catalyzed by the enzyme are presently the subject of some debate. One possibility is the in-line mechanism (53)... 

Stereochemical studies support in-line mechanisms for both the transesterification and hydrolysis steps of ribonuclease catalysis. For example, chiral uridine 2, 3 -cyclic phosphorothioates are hydrolyzed with inversion of configuration, with the diastereoisomer shown yielding a 2 -monophosphothioate of the R configuration at phosphorus. 

A relative of the kinases is adenylate cyclase, whose role in forming the allosteric effector 3, 5 -cyclic AMP (cAMP) was considered in Chapter 11. This enzyme catalyzes a displacement on Pa of ATP by the 3 -hydroxyl group of its ribose ring (see Eq. 11-8, step a). The structure of the active site is known.905 Studies with ATPaS suggest an in-line mechanism resembling that of ribonuclease (step a, Eq. 12-25). However, it is Mg2+ dependent, does not utilize the two-histidine mechanism of ribonuclease A, and involves an aspartate carboxylate as catalytic base.906 All isoforms of adenylate cyclase are activated by the a subunits of some G proteins (Chapter 11). The structures907 of Gsa and of its complex with adenylate kinase905 have been determined. The Gsa activator appears to serve as an allosteric effector. 

In an associative mechanism, there is first the addition of the nucleophile to give a pentacovalent intermediate, followed by the elimination of the leaving group. This mechanism is subdivided further. There is an in-line mechanism, in which the attacking nucleophile enters opposite the leaving group (equation 8.32),... 

The chemistry and stereochemistry of the reactions were extensively discussed in Chapter 8, sections El and E3. There is an in-line mechanism that generates a pentacovalent intermediate or transition state, with the attacking nucleophile and leaving group occupying the apical positions of the trigonal bipyramid. 

Fla. 31. Possible pathways for RNase action. The in-line mechanism is favored for step 1 by Roberts et al. (623), The adjacent mechanism requiring the intermediate step of pseudorotation is suggested by Usher (622) for step 2, the hydrolysis of the cyclic phosphate. 

Since 02 is sterically prevented from attacking opposite 03 and can attack opposite 05" according to the preference rules given in the next section, an in-line mechanism is a simple proposal for step 1. On the other hand, in the hydrolysis the activated OH- attack must occur opposite either 02 or 03 and therefore must also be adjacent to one or the other as illustrated in Fig. 31. Pseudorotation allows such an attack to produce 3 -CMP and Usher s cyclic phosphonate experiments lend support to this hypothesis. 

Roberts et al. (523) emphasized that the in-line mechanism almost requires that two different groups deprotonate the 2 0H and protonate 05 since they are on opposite sides of the basal plane. They are physically removed from each other with a negative P-0- between them to trap any shuttle. Since they observed 3 -CMP interactions with His 12 and 119 by NMR with the greater effect on His 119 and both histidines protonated and since the X-ray structure shows the phosphate between the two histidines, they believed that both histidines are directly involved. Furthermore, the interaction of His 119 is more sensitive to the specific... 

I n this way we have shown that phosphoryl transfer catalysed by Bacillus stearothermophilus and rabbit skeletal muscle phosphofructokinase (6), and rabbit skeletal muscle pyruvate kinase occurs with inversion of configuration at phosphorus (7). The simplest interpretation of these stereochemical results is that phosphoryl transfer occurs by an in-line mechanism in the enzyme substrate ternary complexes. Stereochemical analysis is thus proving to be of considerable importance for delineating the mechanism adopted by phosphokinases. ... 

D-Glycerate-2- and -3-[lsO]phosphorothioates have been synthesized by the route outlined in Fig. 10 [29], Methyl D-glycerate was converted to the diastereomeric mixture of exo- and enr/o-D-glycerate cyclic phosphorothioates, which were separated by chromatography. Each isomer was then subjected to hydrolysis with Lil8OH, which was known to occur largely by an in-line mechanism with inversion of... 

Sp) isomer is cyclized the exo-cyclic phosphorothioate will contain the l80. The labeling pattern follows from the mechanism of this cyclization, specifically by the fact that the base catalyzed internal displacement of the l60- or lsO-activated intermediate proceeds by an in-line mechanism [12,14,31]. 

However, much evidence indicates that enzymes almost always avoid these complexities by using in-line mechanisms. 

Precooling — As discussed in the previous section, it may be necessary to chill the air before entering the adsorbers. This may be accomplished with an in-line mechanical refrigeration unit, a water chiller using evaporative cooling from waste gas from the plant, or a combination of both. 

In the decomposition of 0-phosphobiotin, it is most reasonable to expect that substitution will occur by an adjacent mechanism rather than by an in-line mechanism (53). The attack of bicarbonate on 0-phosphobiotin generates carboxy phosphate, which in turn can decompose to give carbon dioxide, as was discussed previously. If carbon dioxide is to be generated it must be near the N-1 position of biotin with which it must react. In the adjacent mechanism, the car-... 

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