Pyridoxal phosphate resonances

Acid dissociation constants do not tell us which protons dissociate in each step. Assignments for pyridoxal phosphate come from nuclear magnetic resonance spectroscopy [B. Szpoganicz and A. E. Martell, Thermodynamic and Microscopic Equilibrium Constants of Pyridoxal 5 -Phosphate, J. Am. Chem. Soc. 1984,106, 5513]. 

Figure 14-6 Drawing showing pyridoxal phosphate (shaded) and some surrounding protein structure in the active site of cytosolic aspartate aminotransferase. This is the low pH form of the enzyme with an N-protonated Schiff base linkage of lysine 258 to the PLP. The tryptophan 140 ring lies in front of the coenzyme. Several protons, labeled Ha, Hb, and Hd are represented in NMR spectra by distinct resonances whose chemical shifts are sensitive to changes in the active site.169...

Structures of catalytic intermediates in pyridoxal-phosphate-dependent reactions. The initial aldimine intermediate resulting from Schiff s base formation between the coenzyme and the a-amino group of an amino acid (a). This aldimine is converted to the resonance-stabilized... 

Pyridoxal phosphate forms a Schiff base (imine) with the glycine. A carbon-bound hydrogen is labile, and the resulting carbanion stabilized by resonance back into the pyridoxal phosphate. The carbanion approaches the carbonyl carbon of the succinyl-CoA. Following the elimination of the CoASH, the intermediate shown in figure 22.13 is formed. The intermediate then loses a C02, forming a carbanion that is resonance stabilized back into the pyridoxal phosphate. 

The first step involves Schifif base formation by the amino group of the substrate reading with pyridoxal phosphate to form an aldi-mine. This is followed by loss of a functional group (Rg, Equation 17.51), usually by abstraction by an active-site base, to form a resonance-stabilized carbocation. 

The donor amino acid forms a Schiff base with pyridoxal phosphate within the enzyme s active site. After a proton is lost, a carbanion forms and is resonance-stabilized by interconversion to a quinonoid intermediate. After an enzyme-catalyzed proton transfer and a hydrolysis, the a-keto product is released. A second a-keto acid then enters the active site. This acceptor a-keto acid is converted to an a-amino acid product as the mechanism just described is reversed. 

The pyridoxal phosphate-dependent enzymes have been a major focal point in the development of mechanism-based inactivators. Pyridoxal phosphate (PLP) is utilized in resonance stabilization of carbanions at the a- and /3-carbons of amino acids in a variety of reactions which lead to chemical transformations at the a-, /3-, and y-carbons of the substrate (Walsh, 1979). These carbanion equivalents... 

Serine and Glycine. Serine can be converted to glycine by the loss of an active formaldehyde. This reaction is one of the most important suppliers of the Ci fragment. Two coenzymes are necessary, tetrahydrofolate (Chapt. VI-5), and pyridoxal phosphate. The elimination of the /3-C atom is a pyridoxal-catalyzed reaction involving the resonance structure mentioned before in Section 4. While this is taking place, the serine is also bound to tetrahydrofolate. The reaction is reversible Serine is also formed from glycine and active formaldehyde. 

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],... 

The early work of Busby et al. (1975) identified two forms for the enzyme-bound pyridoxal phosphate that could be interconverted by addition of substrate or activator or by phosphorylation. Withers et al. (1979, 1981a) also have identified two slowly exchanging and interconvertable resonances for the pyridoxal phosphate three resonances are sometimes visible in the spectra (Feldmann and Hull, 1977 Hoerl et al, 1979). Although the authors interpreted these as three different environments for the pyridoxal phoq>hate moiety, it cannot be excluded that resonance II is a minor contaminant of Pj or ftw AMP, which would fit with its chemical-shift position and behavior on pH titration. The other two resonances appear at 3.8 i m (form m) and 0.5 ppm (form I), and these also hai pen to be the chemiod shifts observed for the dianionic and monoanionic residues... 

Other enzymes with pyridoxal phosphate moieties have been studied as well. Tryptophanase behaves similarly to cytoplasmic aspartate transaminase, whereas for serine hydroxymethyl transferase the P-NMR resonance is affected by pH but not by ligands (Schnackerz and Bartholmes, 1983 Quashnock et ai, 1983). 

Figure 3-24 The 60-MHz proton magnetic resonance spectrum of pyridoxal 5 -phosphate at neutral pH (apparent pH = 6.65). The internal standard is DSS. Chemical shifts in parts per million are indicated beside the peaks. Spectrum courtesy of John Likos.

The stability of the resonance hybrid (see fig. 10.4/7) accounts for the catalytic action of pyridoxal-5 -phosphate in the reactions shown in equations (3) through (6). 

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