Proline racemase reaction

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

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.

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... 

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... 

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... 

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... 

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