Pyridoxal phosphate enzymes catalytic activity

The chromophoric pyridoxal phosphate coenzyme provides a useful spectrophotometric probe of catalytic events and of conformational changes that occur at the pyridoxal phosphate site of the P subunit and of the aiPi complex. Tryptophan synthase belongs to a class of pyridoxal phosphate enzymes that catalyze /3-replacement and / -elimination reactions.3 The reactions proceed through a series of pyridoxal phosphate-substrate intermediates (Fig. 7.6) that have characteristic spectral properties. Steady-state and rapid kinetic studies of the P subunit and of the aiPi complex in solution have demonstrated the formation and disappearance of these intermediates.73-90 Fig. 7.7 illustrates the use of rapid-scanning stopped-flow UV-visible spectroscopy to investigate the effects of single amino acid substitutions in the a subunit on the rate of reactions of L-serine at the active site of the P subunit.89 Formation of enzyme-substrate intermediates has also been observed with the 012P2 complex in the crystalline state.91 ... 

The addition of cofactors to antibodies is a sure means to confer a catalytic activity to them insofar as this cofactor is responsible for the activity. Indeed for many enzymes, the interaction with cofactors such as thiamins, flavins, pyridoxal phosphate, and ions or metal complexes is absolutely essential for the catalysis. It is thus a question there of building a new biocatalyst with two partners the cofactor responsible for the catalytic activity, and the antibody which binds not only the cofactor but also the substrate that it positions in a specific way one with respect to the other, and can possibly take part in the catalysis thanks to some of its amino acids. 

Suicide Enzyme Inhibitors. Snicide substrates are irreversible enzyme inhibitors that bind covalently. The reactive anchoring group is catalytically activated by the enzyme itself through the enzyme-inhibitor complex. The enzyme thus produces its own inhibitor from an originally inactive compound, and is perceived to commit suicide. To design a substrate, the catalytic mechanism of the enzyme as well as the nature of the functional gronps at the enzyme active site must be known. Conversely, successful inhibition provides valuable information about the structure and mechanism of an enzyme. Componnds that form carbanions are especially usefnl in this regard. Pyridoxal phosphate-dependent enzymes form such carbanions readily becanse... 

In addition to the above reagents, which modify specific tyrosine residues in the protein, desensitization has been reported with pyridoxal phosphate, which forms a Schiff base derivative with lysyl residues (43). This reagent was first reported by Marcus and Hubert (43) to react with FDPase from swine kidney and to abolish AMP sensitivity with very little loss of catalytic activity. With liver FDPase most of the sensitivity to AMP is lost when 7-8 residues are incorporated, with concomitant loss of about 25% of the enzymic activity (43). The effects become irreversible when the Schiff base derivative is reduced with NaBH4 and Are-pyridoxyllysine has been isolated from the reduced complex. In the presence of AMP the sensitive lysine residues are protected, but the amount of PLP incorporated is increased (43). 

Unlike other pyridoxal phosphate-dependent enzymes, in which it is the carbonyl group that is essential for catalysis, the internal Schiff base between pyridoxal phosphate and lysine in glycogen phosphorylase can be reduced with sodium borohydride without affecting catalytic activity. Thus, while pyridoxal phosphate is essential for phosphorylase activity, it does not act by the same kind of mechanism as in amino acid metabolism. 

Studies on the reactivation of apoglycogen phosphorylase with a variety of analogs of pyridoxal phosphate have shown that the catalytic moiety is the 5 -phosphate group - only analogs with a reversibly protonatable dianion in this position have any activity In the nonactivated form of phosphorylase b, the phosphate is monoprotonated (-OPO3H ) when the enzyme has been activated, either allosterically or by phosphorylation (phosphorylase a), it is dianionic (-OPOa ). A glutamate residue in the active site acts as the proton acceptor or donor for this transition between the inactive and active forms of the cofactor. 

Pyridoxal 5 -phosphate (PLP) was noticed to be a constituent of rabbit muscle phosphorylase in 1957, and since that time it has been shown that all a-glucan phosphorylases which give phosphorolysis products with retention of configuration contain PLP. The exact role of the PLP is still not known, though it has been shown that these a-glucan phosphorylases have an absolute requirement for PLP and that the Schiflfbase formed between PLP and glycogen phosphorylase can be reduced with borohydride without eliminating the catalytic activity of the enzyme. The P n.m.r. spectrum of PLP bound to phosphorylase b shows that deprotonation of the 5 -... 

The specific inhibition of D-fructose 1,6-diphosphatase by AMP decreases if the pH of the solution moves399 to above 9. Inhibition by AMP and catalytic activity can be lost by acetylation of the tyrosine residues with 1-acetylimidazole. The presence of substrate or allosteric effectors protects the tyrosine from acetylation.400 Pyridoxal phosphate can also desensitize the enzyme by forming a Schiff base with L-lysinyl residues401 this indicates some participation of L-lysinyl residues in allosteric regulation.401,402... 

As in the case of pyridoxal phosphate, the key to reaction in this case is the use of a heterocyclic compound as an electron sink in the decarboxylation step. Conformational control of the TPP-pyruvate adduct may also be important. The enzyme active site is probably nonpolar, and this provides a significant catalytic factor (112). 

The glutamic acid residue is probably catalytically important because the pH dependence of the rate of inactivation is similar to the pH dependence of V ax with the normal substrates for the enzyme. Moreover, the presence of an active site lysine has been postulated on the basis of pH-rate profiles and on the basis of the loss of activity in the presence of both pyridoxal phosphate and BH4 (99). 

OMP decarboxylase (ODCase) catalyzes the decarboxylation of OMP to UMP, a decarboxylation that must necessarily be mechanistically different from the groups of decarboxylations that occur throughout metabolism [1]. The structure of the substrate does not lend itself to decarboxylation mechanisms involving pyridoxal phosphate (typical of amino acid decarboxylases [2]), thiamine pyrophosphate (typical of a-keto acid decarboxylases [3]), or metal ions (typical of /3-keto acid decarboxylases [4]) although the presence of ions has been detected in some preparations of ODCase [5, 6], the enzyme clearly does not require for catalytic activity [7]. 

Conformational changes have lost some of their mystical associations in recent years. From X-ray crystallography there is now an atomic-level resolution model for both the active, phosphorylated and inactive, dephosphorylated forms of the enzyme glycogen phosphorylase (11). From comparison of these structures, it is seen that the effect of phosphorylation of serine-14 of each subunit is to create an ordered helical conformation at each amino-terminus which in consequence binds more closely to the surface of the glycogen phosphorylase dimer. This produces rotation of each subunit about an axis perpendicular to the axis of symmetry of the dimer. This structural change clearly alters substrate binding at the catalytic site, even though the catalytic pyridoxal phosphates are located more than 30 A from the phosphoserine (12). 

Much of the investigation of the mechanism and catalysis of C=N— forming condensation reactions has been designed to gain insight into the catalytic activity of enzymes which require pyridoxal phosphate (37), for their activity and are involved in the metabolism of amino acids. 

At pH values where the enzyme is bound to pyridoxal as a SchifF base (40), reaction with sodium borohydride destroys the activity of the enzyme by reducing the SchifF base to a pyridoxylamine (42). This observation gives strength to the hypothesis that the subsequent reaction of enzyme with an amino acid involves transamination. Skeletal muscle phosphorylase is an exception , indicating that this particular enzyme does not require a SchifF base linkage for its activity. Further research is required to determine whether the role of pyridoxal phosphate in this enzyme can be attributed simply to maintenance of the active site of the enzyme in the required conformation or whether a new set of catalytic properties must be ascribed to the vitamin Bg aldehyde. 

Kynureninase (Figure 11.16) is a pyridoxal phosphate-dependent enzyme, and its activity falls markedly in vitamin deficiency, at least partly because it undergoes a slow mechanism-dependent inactivation that leaves catalytically inactive pyridoxamine phosphate at the active site of the enzyme. The enzyme can only be reactivated if there is an adequate supply of pyridoxal phosphate. This means that in vitamin deficiency there is a considerable accumulation of both hydroxykynurenine and kynurenine, sufficient to permit greater metabolic flux than usual through kynurenine transaminase, resulting in increased formation of kynurenic and xanthurenic acids. 

The oxidative pathway of tryptophan metabolism is shown in Figure 3. Kynureninase is a pyridoxal phosphate-dependent enzyme, and in deficiency its activity is lower than that of tryptophan dioxygenase, so that there is an accumulation of hydroxy-kynurenine and kynurenine, resulting in greater metabolic flux through kynurenine transaminase and increased formation of kynurenic and xanthurenic acids. Kynureninase is exquisitely sensitive to vitamin Bg deficiency because it undergoes a slow inactivation as a result of catalysing the half-reaction of transamination instead of its normal reaction. The resultant enzyme with pyridoxamine phosphate at the catalytic site is catalytically inactive and can only be reactivated if there is an adequate concentration of pyridoxal phosphate to displace the pyridoxamine phosphate. 

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