Mandelate racemase. V. Mandelate racemase from Pseudomonas putida. Magnetic resonance and kinetic studies of the mechanism of catalysis

Abstract

The interactions of mandelate racemase with divalent metal ion, substrate, and competitive inhibitors were investigated. The enzyme was found by electron paramagnetic resonance (EPR) to bind 0.9 Mn2+ ion per subunit with a dissociation constant of 8 muM, in agreement with its kinetically determined activator constant. Also, six additional Mn2+ ions were found to bind to the enzyme, much more weakly, with a dissociation constant of 1.5 mM. Binding to the enzyme at the tight site enhances the effect of Mn2+ on the longitudinal relaxation rate (1/T1p) of water protons by a factor of 11.9 at 24.3 MHz. From the frequency dependence of 1/T1p, it was determined that there are similar to 3 water ligands on enzyme-bound Mn2+ which exchange at a rate larger than or equal to 10-7 sec-1. The correlation time for enzyme-bound Mn2+-water interaction is frequency-dependent, indicating it to be dominated by the electron spin relaxation time of Mn2+. Formation of the ternary enzyme-Mn2+-mandelate complex decreases the number of fast exchanging water ligands by similar to 1, but does not affect tau-c, suggesting the displacement or occlusion of a water ligand. The competitive inhibitors D,L-alpha-phenylglycerate and salicylate produce little or no change in the enzyme-Mn2+-H2O interaction, but ternary complexes are detected indirectly by changes in the dissociation constant of the enzyme-Mn2+ complex and by mutual competition experiments. In all cases the dissociation constants of substrates and competitive inhibitors from ternary complexes determined by magnetic resonance titrations agree with K-M and K-i values determined kinetically and therefore reflect kinetically active complexes. From the paramagnetic effects of Mn2+ on 1/T1 and 1/T2 of the 13C-enriched carbons of 1-[13C]-D,L-mandelate and 2-[13C]-D,L-mandelate, Mn2+ to carboxylate carbon and Mn2+ to carbinol carbon distances of 2.93 plus or minus 0.04 and 2.71 plus or minus 0.04 A, respectively, were calculated, indicating bidentate chelation in the binary Mn2+-mandelate complex. In the active ternary complex of enzyme, Mn2+, and D,L-mandelate, these distances increase to 5.5 plus or minus 0.2 and 7.2 plus or minus 0.2 A, respectively, indicating the presence of at least 98.9% of a second sphere complex in which Mn2+, and C1 and C2 carbon atoms are in a linear array. The water relaxation data suggest that a water ligand is immobilized between the enzyme-bound Mn2+ and the carboxylate of the bound substrate. This intervening water ligand may polarize or protonate the carboxyl group. From 1/T2p the rate of dissociation of the substrate from this ternary complex (larger than or equal to 5.6 times 10-4 sec-1) is at least 52 times greater than the maximal turnover number of the enzyme (1070 sec-1), indicating that the complex detected by nuclear magnetic resonance (NMR) is kinetically competent to participate in catalysis. Relationships among the microscopic rate constants are considered.

Detailed Reaction Mechanism of Macrophomate Synthase

“…The tight binding of Mg(II) and the high specificity for Mg(II) are relatively unusual in metal-dependent ␤-ketoacid decarboxylases such as malic enzyme (18) and oxalacetate decarboxylase from Pseudomonas putida (19) and Acetobacter xylinum (20), in which both Mg(II) and Mn(II) are equally effective in their catalysis. The K d is at least 20 times lower than those for the enolase superfamily (21), mandelate racemase (8 M) (22) and enolase (1.8 M) (23), which possess relatively higher affinity for divalent metals. These enzymes do not show any predominant specificity for divalent metal ions.…”

The Substrate Spectrum of Mandelate Racemase: Minimum Structural Requirements for Substrates and Substrate Model

“…[26] Racemization takes place via deprotonation by two enantiomer-specific bases juxtaposed on either side of the chiral a-carbon atom, i.e., His-297 and Lys-166 for (R)-and (S)-mandelate, respectively; while one base acts as base, the other functions as the corresponding acid for proton delivery from the opposite side. This "molecular ping-pong game" makes mandelate racemase an extremely efficient catalyst with a turnover frequency of 1000 s À 1 , [27] meaning that 1.0 g of the enzyme racemizes approximately 1.7 kg of mandelic acid per hour.…”

“…All attempts to alter the a-hydroxy group failed so far: a-phenylglycine (30) and the corresponding N-acetyl derivative (31) were totally inactive, most likely due to the inability of the amino/ammonium or aminoacetyl group to form a tight salt bridge with the catalytically active Mg 2 þ ion. Most remarkably, variation of the carboxylate moiety to the corresponding carboxamide was tolerated to some extent: mandeloamide (27) and its p-bromo analogue (28) were racemized with acceptable rates (15 and 22%, respectively).…”

Enzymatic Racemisation and its Application to Synthetic Biotransformations

“…, [19] meaning 1.0 g of the enzyme racemises approximately 1.7 kg of mandelic acid per hour. Electron-withdrawing substituents in the paraposition, such as p-Cl and p-Br in mandelic acid derivatives of type 4 or p-Br in amide 3 increase the activity of mandelate racemase, while electron-donating substituent (p-OH, p-OMe) decrease the activity; metaderivatives, like m-Cl-4, are racemised at a lower rate compared to the para-analogues, which might be due to steric hindrance.…”