Mandelate racemase active site

Figure 17-19. Models of the mandelate racemase active site with complexed substrate, p-iodomandelate. Reprinted from Neidhartet al.11941.

Other Proteins The ouabain-binding site on (Na /K -adenosine-5 -triphosphatase, 46, 523 penicillin isocyanates for /3-lactamase, 46, 531 active site-directed addition of a small group to an enzyme the ethylation of ludferin, 46, 537 mandelate racemase, 46, 541 d imethylpyrazole carboxamidine and related derivatives, 46, 548 labeling of catechol O-methyltransferase with N-haloace-tyl derivatives, 46, 554 affinity labeling of binding sites in proteins by sensitized photooxidation, 46, 561 bromocolchicine as a iabei for tubuiin, 46, 567. 

Figure 13-5 An S-mandelate ion in the active site of mandelate racemase. Only some of the polar groups surrounding the active site are shown. The enzyme has two catalytic acid-base groups. Lysine 166 is thought to deprotonate S-mandelate to form the aci anion, while His 297 deprotonates R-mandelate to form the same anion.106...

P. C. Babbitt, J. A. Gerlt, G. A. Petsko, and D. Ringe, Evolution of an enzyme active site the structure of a new crystal form of muconate lactonizing enzyme compared with mandelate racemase and enolase, Proc. Natl. Acad. Sci. USA 1998,... 

X 10 if the enolate anion intermediate were not stabilized in the active site this value is 10 -fold less than the observed value for the kcat, 500 s. Recall that an enolate anion is necessarily on the reaction coordinate, so the value of AG° must be reduced for the enolate anion to be kinetically competent irrespective of whether AG int can be reduced. Thus, the active site of mandelate racemase must decrease AG° from the value predicted from the values of the substrate carbon acid and the active site base in solution. The obvious strategy to accomplish this reduction is preferential stabilization of the enolate anion intermediate relative to the carbon acid substrate, the increased negative charge on (or proton affinity of) the carbonyl/carboxylate oxygen of the enolate anion intermediate provides a convenient handle for enhanced electrostatic or hydrogen bonding interactions with the active site. 

Both the 1,1-proton transfer reaction catalyzed by mandelate racemase (MR) and the dehydration catalyzed by enolase require Mg + for activity. The values of the pK s for mandelate and 2-phosphoglycerate, the substrates for the MR- and enolase-catalyzed reactions, are estimated as 29 and 32, respectively [1]. The values of the pKaS of the general basic Lys residues are 6 and 9 in MR [6] and enolase [73], respectively. Thus, formation of a dienolate anion intermediate is extremely endergonic, unless the active site can stabilize the intermediate which is the obvious function of the essential Mg. The rate accelerations for the MR- and enolase-catalyzed reactions are 10 as a direct result of the values of the pKaS of the a-protons (Table 6.1). 

Figure 7.13. Active site of the K166R mutant of mandelate racemase from the costal structure of the Michaelis complex with (R)-mandelate. All distances are in A [51],...

The enzyme mandelate racemase from the soil bacterium Pseudomonas putida catalyzes the racemization of the (/ )- and (5)-enantiomers of mandelate, as shown in Figure 3. In the action of the enzyme a hydrogen atom (a-proton) is extracted from the aliphatic carbon atom of the mandelate ion. Two active-site bases are involved in this one to extract the a-proton from the (5)-isomer and the other to extract the a-proton from the (R)-isomer [26]. A proton from a solvent molecule in the active site then adds to the deprotonated mandelate in one or other of the two... 

Compared to similar cases, however, NH OH-induced release of label from mandelate racemase appears to be rather slow. This may mean either that the active site of blocked racemase is relatively inaccessible to small molecules such as NH2OH or that the a-phenyglycidate alkylates more than one type of amino acid residue. ... 

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