Pyridoxal 5 -phosphate hydrogen bonding

Biotin (5) is the coenzyme of the carboxylases. Like pyridoxal phosphate, it has an amide-type bond via the carboxyl group with a lysine residue of the carboxylase. This bond is catalyzed by a specific enzyme. Using ATP, biotin reacts with hydrogen carbonate (HCOa ) to form N-carboxybiotin. From this activated form, carbon dioxide (CO2) is then transferred to other molecules, into which a carboxyl group is introduced in this way. Examples of biotindependent reactions of this type include the formation of oxaloacetic acid from pyruvate (see p. 154) and the synthesis of malonyl-CoA from acetyl-CoA (see p. 162). 

Kim, Chin, and co-workers have described a highly interesting oxyanion hole mimic that transforms L-amino acids to D-amino acids [97]. The mechanism involves stabilization of the enolate intermediate by an internal hydrogen bond array generated by urea group (Scheme 4.14). In the presence of an external base, such as triethylamine, the receptors readily promote the epimerization of a-amino acids, favoring the D-amino acids due to unfavorable steric interactions in the receptor-L-amino acid complex. These receptors can also be viewed as chiral mimics of pyridoxal phosphate [98]. 

Figure 3-30 Spectra of the pyridoxal phosphate (PLP), pyridoxamine phosphate (PMP) and apoenzyme forms of pig cytosolic aspartate aminotransferase at pH 8.3, 21 °C. Some excess apoenzyme is present in the sample of the PMP form. Spectra were recorded at 500 MH2. Chemical shift values are in parts per million relative to that of HzO taken as 4.80 ppm at 22°C. Peak A is from a proton on the ring nitrogen of PLP or PMP, peaks B and D are from imidazole NH groups of histidines 143 and 189 (see Fig. 14-6), and peaks C and D are from amide NH groups hydrogen bonded to carboxyl groups.

Except for some vitamin B12-dependent reactions, the cleavage or formation of carbon-carbon bonds usually depends upon the participation of carbonyl groups. For this reason, carbonyl groups have a central mechanistic role in biosynthesis. The activation of hydrogen atoms (3 to carbonyl groups permits (3 condensations to occur during biosynthesis. Aldol or Claisen condensations require the participation of two carbonyl compounds. Carbonyl compounds are also essential to thiamin diphosphate-dependent condensations and the aldehyde pyridoxal phosphate is needed for most C-C bond cleavage or formation within amino acids. 

The coenzyme, pyridoxal 5 -phosphate (PLP), forms an aldimine linkage with the -amino group of Lys-258 via its aldehyde group. PLP also interacts with Asp-222, Tyr-225 and Asn-194 by forming hydrogen bonds thus it maintains proper orientation at the active site. The enzyme is in the open conformation. 

Fig. 6.2 Spatial structure of the active site of rabbit muscle phosphorylase85 (A) and sequence comparison of the active site residues (B). A, Hydrogen bonds of less than 3.3 A (dotted lines) and water molecules (crosses) are also indicated. B, Sequences constituting the active-site region are compared among potato type-H isozyme (H), type-L isozyme (L), and rabbit muscle enzyme (R). Residues making van der Waals contact with the pyridoxal moiety and the 5 -phosphate group are boxed. (From J. Biol. Chem., 261 (18), 8233 (1986)).

Because pyridoxal phosphate is bound to lysine in this way, the resolution of holoenzymes to yield the apoenzyme is difficult unless this Schiff base can be reacted with a carbonyl-trapping reagent to give an adduct that can be removed by dialysis. The pyridoxamine phosphate form of aminotransferases (Section 9.3.1.3) can be resolved more readily, because the coenzyme is only held by ionic bonds to the 5 -phosphate, hydrophobic interactions with the 2 -methyl group, and hydrogen bonding to the heterocyclic nitrogen. 

The pyridoxal phosphate complex of aminoacrylic acid can also be formed from serine by loss of an OH radical in a manner analogous to loss of the. 4.r" radical depicted above. This complex contains a reactive double bond to which the reactive /3-hydrogen of indole can add, giving a complex which on hydrolysis yields tryptophan. Such a mechanism is in accord with the known facts on tryptophan biosynthesis (cf. 858, and previous discussion, p. 41). 

Although pyridoxal phosphate is involved in a wide variety of reactions the processes are all related mechanistically by the electron withdrawal toward the cationic or hydrogen-bonded imine nitrogen and into the electron sink of the pyridine/pyridinium ring. 

Tong, H., and Davis, L. (1995). 2-amino-3-ketobutyrate-CoA ligase from beef liver mitodion-dria An NMR spectroscopic study of low-barrier hydrogen bonds of a pyridoxal 5 -phosphate-dependent enzyme. Biochemistry 34, 3362-3367. 

Spectral studies show that in the acid pH range, pyridoxal phosphate is bound to the protein as a Schiff base (40) and infrared evidence suggests that there is a strong hydrogen bond between the phenolic hydroxyl group and the imino nitrogen atom . At higher pH values... 

The electron displacement thus induced with its consequent weakening of the bonds to the a-hydrogen, the /3-carbon, and the carboxyl groups of the amino acid is the essential feature of the activation reaction of amino acids by pyridoxal phosphate-dependent enzymes. 

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