Enzyme proline racemase

Fig. 22 The free energy diagram for proline racemase, showing the effect on the free energy of the enzyme of increasing substrate concentration c. When c < cD, the system is unsaturated. When cD < c < cP, the system is saturated and when c > cP, the system is oversaturated, with transition states 7 and 8 rate-limiting.

Figure 3. Computed three-dimensional free energy diagram for proline racemase x-axis, the reaction coordinate y-axis, free energy and z-axis, substrate saturation. At the front of the diagram, [S] = [P] = 1 /aM, where the enzyme is unsaturated at the back of the diagram, [S] = [P] = 1 M, where the enzyme is oversaturated. Reproduced from reference 9 with permission of the authors and the American Chemical Society.

TRIOSE-PHOSPHATE ISOMERASE AFFINITY LABELING HALDANE RELATION ENZYME ENERGETICS (The Case of Proline Racemase)... 

Strain and stress in enzymes arise from several different causes. We have seen in this chapter, and we shall see further in Chapters 15 and 16, that stress and strain may be divided into two processes, substrate destabilization and transition state stabilization. Substrate destabilization may consist of steric strain, where there are unfavorable interactions between the enzyme and the substrate (e.g., with proline racemase, lysozyme) desolvation of the enzyme (e.g., by displacement of two bound water molecules from the carboxylate of Asp-52 of lysozyme) and desolvation of the substrate (e.g., by displacement of any bound water molecules from a peptide28). Transition state stabilization may consist of the presence of transition state binding modes that are not available for the... 

In a study of the highly purified alanine racemase of E. coli, Lambert and Neuhaus determined significant differences in the maximal velocities and the Michaelis-Menten constants of the substrates in the forward (L - dl) and reverse directions (d - dl) [37]. From these data the value calculated for Keq is 1.11 0.15. The time course of the reaction showed that in 10 min with L-alanine as substrate ca. 0.09 jumol of D-alanine were formed. With the same amount of enzyme (750 ng) and in the same time period, ca. 0.05 jamol of L-alanine were formed from D-alanine. Similar results have been reported for the same enzyme from S. faecalis and for proline racemases [37]. Thus, in these cases, there are definite kinetic differences, as expected for the existence of two diastereoisomers formed between enzyme and two substrate enantiomers. 

We turn now to compounds that provide the most intimate views of the catalytic process itself Linus Pauling proposed in 1948 that compounds resembling the transition state of a catalyzed reaction should be very effective inhibitors of enzymes. These mimics are called transition-state analogs. The inhibition of proline racemase is an instructive example. The racemization of proline proceeds through a transition state in which the tetrahedral a- carbon atom has become trigonal by loss of a proton (Figure 8.24). In the trigonal form, all three bonds are in the same plane C also carries a... 

Figure 8.24. Inhibition by Transition State Analogs. (A) The isomerization of 1-proline to d-proline by proline racemase, a bacterial enzyme, proceeds through a planar transition state in which the a carbon is trigonal rather than tetrahedral. (B) Pyrrole 2-carboxylate, a transition state analog because of its trigonal geometry, is a potent inhibitor of proline racemase.

Slow proton transfer makes possible the occurrence of iso mechanisms -mechanisms in which the form of the enzyme released after catalysis is different to that at the start of the cycle. A candidate would be any inverting glycosidase, which is released with the acid group deprotonated and the basic group protonated [Figure 1(b)], although no example in the glycosyl transfer area has yet been demonstrated (the best example is proline racemase, " in which two cysteines act, one as an acid and the other as a base). 

Proline racemase is a member of a broad family of cofactor-independent epimerases and racemases, and has been very well characterized mechanistically. The proline racemase from Clostridium sticklandii was the first of the cofactor-independent racemases to be characterized [13, 80], The enzyme participates in the catabolism of L-proline, producing o-proline as a substrate for o-proline oxidase [4]. Early... 

Figure 7.16. Mechanism for the stereoinversion of L- to D-proline catalyzed by proline racemase (upper manifold) and water catalyzed proton exchange of the free enzyme (lower manifold).

Rudnick and Abeles purified proline racemase to 95% homogeneity from Clostridium sticklandii, and characterized it 92. The enzyme is composed of two identical subunits with a molecular weight of about 38000, and is independent of any cofactors or metals. Most amino acid racemases require pyridoxal 5 -phosphate, which labilizes the bond between the a-hydrogen and the chiral center by aldimine formation with the a-amino group of the substrate. However, PLP is not involved in the reaction of proline racemase acting on an a-imino acid. The enzyme also acts on 2-hydroxy-L-proline and 2-allo-hydroxy-D-proline although slowly they are epimer-ized at a rate of 2 and 5% of the rate of L-proline racemization, respectively. L-Proline and D-proline showed Km values of 2.9 and 2.5 mti, respectively1119. ... 

A racemase is a difficult enzyme with which to study product inhibition, since Teq = I and, if one puts in enough product to inhibit the enzyme, one is near equilibrium or the reaction goes backward. In their epic studies on proline racemase, Knowles and co-workers solved this problem by using optical rotation to... 

The countertransport or tracer perturbation method, introduced by Britton and Clarke (66), involves a test for obligate free enzyme isomerization (i.e., an Iso mechanism). The method was used by Knowles and co-workers to prove the Iso mechanism for proline racemase and show that V is limited by EA to E F, rather than by E to E, conversion (67). 

Interestingly, proline racemase showed no countertransport in ammonium bicarbonate buffer, as the buffer ions catalyze the E to E conversion so that it no longer can be made rate limiting (67). The E to E conversion involves the deprotonation of a water (or small buffer) molecule in the active site to give hydroxide (or deprotonated buffer) by one base, and the subsequent protonation of hydroxide (or buffer) by the other base. Thus, one of the bases on the enzyme (which are sulfhydryl groups) must be protonated and the other unprotonated for activity, and which one is protonated determines whether L- or D-proline is adsorbed 68). 

The iso mechanisms, with isomerization of a stable enzyme form, are not very common in ordinary soluble enzyme systems. Well characterized examples of iso mechanisms are proline racemase, funiarase, and carbonic anhydrase (Rebholz Northrop, 1995). 

Yamauchi et al.11121 concluded that aspartate racemase also uses two bases to remove and return the a-proton of the substrate. Aspartate racemase contains three cysteine residues Cys 84, Cys 190 and Cys 197, and only Cys 84 is essential for the enzyme activity. The alkylation of one cysteine residue/dimer with 2-nitro-5-thiocya-nobenzoic acid results in a complete loss of activity. Therefore, the enzyme shows a half-of-the-sites-reactivity11121. Yamauchi et al.11121 suggested that the enzyme has a composite active site formed at the interface of two identical subunits in the same manner as proposed for proline racemase1921. 

A variety of other enzymes involved in amino acid catabolism have been detected in both protozoa and helminths. These include deaminases such as histidase, decarboxylases, some of which are involved in biosynthesis of amines and related compounds, and hydroxylases of proline, tryptophan and tyrosine. These additional enzymes have mostly been reported in helminths (1). L-Amino acid oxidases and D-amino acid oxidases are also present and the availability of the latter would allow D-amino acids to be metabolized in the absence of amino acid racemases. 

A planar analog of proline is pyrrole 2-carboxylate, and this is a potent inhibitor of the racemase. It gives rise to 50% inhibition at a concentration 160 times less than the concentration of D- or L-proline that gives 50% saturation of binding. Therefore it is an excellent example of a transition-state analog of the enzyme. 

Other amino acids such as L-aspartate and L-serine, can spontaneously racemize with aging in humans [149]. It has been reported that microwaving milk racemizes L-proline to D-proline, which could reduce the milk s nutritional value [150]. For L-serine, a serine racemase has been characterized that catalyzes the direct racemization of L-serine to D-serine. This enzyme can also catalyse D-serine to L-serine but with lesser affinity. As a number... 

Racemases are enzymes that catalyze the inversion of the chiral center by deprotonation of the C , followed by reprotonation on the opposite face of the planar carban-ionic transition-state species [13,14], In order to overcome the high energetic barrier of racemization, for example, on a-amino acids, some racemases employ pyridoxal phosphate (PLP) as a cofactor to use the resonance-stabilized amino acid complex as an electron sink because the estimated pK values for the C of amino acids are high, in the range 21-32 [14,15]. The formation of an imine PLP-substrate covalent bond makes the pK value of a-hydrogen of amino acids low. The second class of enzymes includes proline, aspartate, and glutamate racemases and diaminopimelate epimer-ase, with a cofactor-independent two-base mechanism [14],... 

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