Proline racemase and

Proline racemase occurs in Clostridium sticklandii, which produces 8-aminovalerate from L-proline. Proline racemase and D-proline reductase are responsible for the conversion L-proline is racemized by proline racemase to form D-proline, which is converted into 5-aminovalerate by D-proline reductase (E.C. 1.4.4.1). 

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). 

In the experiment, 33 mM labeled DL-proline and 183 mA unlabeled L-proline were incubated with proline racemase and the percent label in D- and L-proline monitored as L-proline was converted to D-proline and the reaction approached chemical and isotopic equilibrium. The percent label in L-proline rose to 65%, then gradually dropped to 50%, showing that there was countertransport of label from D- to L-proline induced by the high flux from l- to D-proline. This countertransport results from the high level of E formed by the reaction E -I- A F -I- E, which increases the rate of the reverse reaction, F -(- E E -I- A. The fractional excess of label in A at any point during the reaction is given by... 

FIGURE 16.7 The proline racemase reaction. Pyrrole-2-carboxylate and A-l-pyrroline-2-carboxylate mimic the planar transition state of the reaction. 

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.

Stadtman TC, Elliott P. 1957. Studies on the Enzymatic Reduction of amino acids II. Purification and properties of ao-proline reductase and a prohne racemase from Clostridium sticklandii. 1 Biol Chem 228 983-97. 

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.

A number of other racemases and epimerases may function by similar mechanisms. While some amino acid racemases depend upon pyridoxal phosphate (Chapter 14), several others function without this coenzyme. These include racemases for aspartate,113 glutamate,114-1153 proline, phenylalanine,116 and diamino-pimelate epimerase.117 Some spiders are able to interconvert d and l forms of amino acid residues in intact polypeptide chains.118119... 

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. 

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). 

As first espoused by Knowles and Albery, the limiting selective pressure on enzymatic function is the diffusion-controlled limit by which substrates bind and products dissociate [7]. In the case of triose phosphate isomerase [8], ketosteroid iso-merase [9], mandelate racemase [10], and proline racemase [11], the energies of various transition states on the reactions coordinates have been quantitated, with the result that the free energies of the transition states for the proton transfer reactions to and from carbon are competitive with those for substrate association/ product dissociation. However, as discussed in later sections, the energies of the... 

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).

Isotope discrimination studies were employed to deduce if the double proton transfer of proline racemase is concerted or stepwise [88]. Isotope discrimination is an alternative manifestation of the multiple kinetic isotope effect techniques previously discussed, wherein racemization is conducted in mixed isotopic solvents of H2O and D2O and the discrimination in the incorporation of solvent deuterium is measured. If the double proton transfer is stepwise, deuteration of the substrate... 

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