Internal aldimine

ACS isozyme utilizes pyridoxal-5 -phosphate (PLP) as a cofactor and belongs to fold type I PLP-dependent enzymes showing an absorption maximum between 422 and 431 nm, which is due to the internal aldimine. The reaction mechanism proposed for the conversion of SAM to ACC by ACS illustrated in Scheme 2 involves the following steps ... 

The active site lysine of ACS forms a Schiff base (internal aldimine) via its e-amino group with the bound PLP in the unliganded enzyme (Scheme 2(a)). 

D). The coenzyme is shown as the internal aldimine with Lys 258 (see Fig. 14-6,14-10). The positive and negative contours on the two sides of the coenzyme ring indicate that the coenzyme tilts over to form the external aldimine when substrates react.413 (D) Superimposed structure of the active site of the enzyme in its free form as in (A) (bold lines) and the refined structure of the a-methylaspartate complex, (dashed lines).411 This illustrates the tilting of the coenzyme ring, which is also shown in Eq. 14-39 and Fig. 14-10. Courtesy of Arthur Amone and Sangkee Rhee. 

Before discussing the reactions of Schiff bases of PLP we should consider one fact that was not known in 1952. PLP is bound into an enzyme s active site as a Schiff base with a specific lysine side chain before a substate binds. This is often called the internal aldimine. When the substrate binds it reacts with the internal Schiff base by a two-step process called transimination (Eq. 14-26) to form the substrate Schiff base, which is also called the external aldimine. 

In the active site of the enzyme PLP forms an internal aldimine (Schiff base) with Lys-270 (Fig. 9.13,1). When the substrate is bound at the active site, its a-amino group attacks the C-4 atom of the coenzyme and replaces the -amino group of Lys-270 from its bond with PLP. This transaldimination reaction probably proceeds via a tetrahedral intermediate (a gem-diamine). Spectral evidence for formation of a gem-diamine in this step has recently been obtained in studies of the reaction of tryptophanase with L-homophenylalanine.41 The gem-diamine is subsequently converted to an external, PLP-substrate aldimine, and the -amino group of Lys-270 is released (Fig. 9.13, II). The equilibrium constant of this step with L-tryptophan is determined to be 11.6 mM.78 ... 

The a -amino group of the amino acid substrate displaces the e-amino group of the active-site lysine residue. In other words, an internal aldimine becomes an external aldimine. The amino acid-PLP Schiff base that is formed remains tightly bound to the enzyme by multiple noncovalent interactions. 

Figure 24.9. Amino Acid Biosynthesis by Transamination. Within a transaminase, the internal aldimine is converted into pyridoxamine phosphate (PMP) by reaction with glutamate. PMP then reacts with an a-ketoacid to generate a ketimine. This intermediate is converted into a quinonoid intermediate, which in turn yields an external aldimine. The aldimine is cleaved to release the newly formed amino acid to complete the cycle.

We shall review the transaminase mechanism (p. 657) as it applies to amino acid biosynthesis (see Figure 23.11). The reaction pathway begins with pyyi-doxal phosphate in a Schiff-base linkage with lysine at the transaminase active site, forming an internal aldimine (Figure 24.7). An amino group is... 

Fig. 1. (continued) (h) Pantothenate, (i) Pyridoxine, (j) Pyridoxal 5 -phosphate, (k) pyridoamine 5 -phosphate, (I) Schiff base between PLP and lysine in the active site forming the internal aldimine, (m) Thiamine pyrophosphate, (n) Iron-porphirin, (o) Protoporphirin IX,... 

This enzyme represents an interesting contrast to tryptophan synthase, which catalyzes the essentially irreversible formation of i-Trp. The spectrum of the native enzyme, which is highly pH dependent, is characterized by two absorbance bands centered at 420 nm and 337 nm. Early RSSF investigations utilizing rapid incremental jumps in pH showed that the two spectral bands arise from different protonation states of the covalently bound internal aldimine, E(Ain), form of the cofactor (101). Studies with a variety of amino acid inhibitors of tryptophanase (amino acids, which react reversibly with the enzyme to form covalent PLP-intermediates, but cannot complete the P-elimination reaction to form products), showed that the 420-nm species is the reactive form of the cofactor. The 337-nm species must be converted to the 420-nm species before reaction with the amino group of the substrate will occur. The 420-nm species represents aketoenamine form of the cofactor in which the iminium nitrogen of the Schiff s base is protonated (102). 

Figure 17-1. Mechanism of the alanine racemase reaction. A, An internal aldimine of PLP with a lysyl residue B, an external aldimine of PLP with D-alanine C, a quinonoid intermediate formed after removal of a hydrogen from alanyl external aldimines B or D D, an external aldimine of PLP with L-alanine. Reprinted from Watanabe et al.[331.

Figure 2 Crystal structure of E. coli PdxF (SerC) (PDB code 1 bjo). (a) An illustration of the homodimer with bound ligands. Both subunits have PLP bound, one as an external aldimine with the analogue a-methyl-L-glutamate, and the other as an internal aldimine with K198. (b) The active site containing the external aldimine identifying residues within 5 A of a-methyl-L-glutamate. Three residues (R42, H41, and T240) are from the other subunit and denoted by green text.

Figure 18 The two haif reactions of the transamination process. The cofactor (Py) shuttles between the pyridoxal phosphate form (Py-CHO), bound to the enzyme as internal aldimine, and the pyridoxamine phosphate (PM P) form (Py-NHz) ...

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