CATALYSIS − Rakyat Biologi

SR catalyses the conversion of L- to D-serine and vice versa exploiting the electron withdrawal capacity of PLP. In PLP-dependent enzymes the carbanion formed on the substrate by removal of an a-substituent (for example, the proton or the carboxylate) is stabilized by PLP, that acts as an “electron sink” and delocalizes the negative charge on the pyridine ring (45). PLP-dependent enzymes are widely distributed among organisms and perform most of the reactions of amino acids metabolism (racemization, decarboxylation, transamination) (46). PLP-dependent enzymes are known for their promiscuity, i. e. the same enzyme catalyses more than one reaction on the substrate and/or more than one substrate can be processed by the same enzyme. SR is no exception and in fact can perform b-elimination as well on serine and several amino acid derivatives. In general, racemisation requires the abstraction of the a-proton and reprotonation of C_a on the opposite face, with formation of the antipodal amino acid. The mechanism of PLP-dependent racemization of amino acids has been investigated in detail on bacterial enzymes (1), especially alanine racemase due to its importance as a potential antibiotic target (47-49).

Due to the low expression yields, the reaction mechanism of mammalian SR has never been studied in detail and has mostly been inferred from data on the bacterial enzymes and from structural information (17, 18). The incoming amino acid, either L- or D-serine with an unprotonated a-amino group, attacks the C_4’ of the Schiff base of the internal aldimine (Intermediate 1) to form the external aldimine (Intermediate 2) with release of unprotonated Lys56. Racemisation proceeds via a two-base mechanism, with Lys56 and Ser84 acting on the opposite sides of the amino acid. In this mechanism, D- and L-serine are deprotonated by distinct residues (L-Ser by Lys56 and D-Ser by Ser84) and reprotonated on the opposite face of the carbanion by the other one (L-Ser by Ser84 and D-Ser by Lys56). It has been pointed out that this mechanism should reduce the chance for competing reactions to take place, because the acid is pre-positioned to quickly reprotonate the intermediate (47). In fold-type I or IV enzymes the pyridine nitrogen is protonated and the positive charge is stabilized by a conserved negatively charged residue. The functional role of this residue is to increase the electron sink potential of the pyridine ring and thus to further stabilize the carbanionic intermediate. This feature is not present in fold-type II enzymes, where a polar/neutral amino acid is always facing the pyridine nitrogen that is consequently unprotonated (18). For this reason some authors proposed that no quinonoid intermediate (Intermediate 3) forms during catalysis by SR and the reaction proceeds with formation of a true carbanion with no delocalization of the negative charge to the pyridine ring (18). However, for alanine racemase, it was suggested that formation of a rather unstable quinonoid intermediate helps in controlling reaction specificity by kinetically preventing the conformational changes needed for transamination (47). Further investigations are needed to establish if this is also the case for SR in view of the relative efficiency of the competing b-elimination reaction.

All hSR orthologs characterized to date show some degree of b-elimination, with a relative efficiency with respect to racemisation that decreases going from archea to mammals (12). This observation, together with the finding that serine/threonine dehydratases are the SR closest relatives, suggests that b-elimination could be a residual activity from ancestral enzymes. The first experimental evidence that SR is able to perform b-elimination came from studies on b-substituted amino acids (50). In particular, b-elimination of L-serine-O-sulfate is 500 times faster than L-serine racemisation. However, b-elimination on serine was not detected until ATP and Mg⁺⁺ were discovered to be essential cofactors (34, 35). Elimination proceeds with the protonation of the quinonoid intermediate on the b-hydroxy group that is subsequently eliminated as water to form the a-aminoacrylate (17, 18) (Figure 3). Protonation of the b-hydroxy group requires a strong acid. In the case of mammalian serine dehydratase the phosphate group of PLP acts as acid (27). It was proposed that also for SpSR the phosphate group is in the correct orientation to donate a proton to the leaving b-hydroxy group (18). However, hSR seems to work by a different mechanism, where the catalytic Lys56 or Ser84 function as acids to protonate the hydroxyl group of L- and D-serine, respectively (17). Noticeably, functional data show a lower efficiency for D-serine elimination with respect to L-serine. This difference is bigger for the human than the mouse enzyme (22, 51). The a-aminoacrylate is unstable and after release is rapidly hydrolysed to pyruvate and ammonia. The a-aminoacrylate Schiff base is an extremely reactive intermediate that undergoes covalent modifications due to side reactions in PLP-dependent enzymes (52, 53). These syncatalytic modifications often result in the inactivation of the enzyme. Syncatalytic reactions following a-aminoacrylate formation have never been reported for hSR (54), although in the yeast S. pombe, formation of a-aminoacrylate is followed by conversion to PLP-lysino-D-alanine with retention of 50-60 % of the initial activity (18, 19).

Whereas SR shows a high substrate specificity for racemisation, that is restricted to serine (55), b-elimination has a broader substrate specificity and is effectively carried out on L-and D-serine, L-threonine, L-Ser-O-sulfate, L-threo-3-hydroxyaspartate and b-chloro-L-alanine (7, 22, 34, 50, 55). The elimination is a reaction conserved through evolution (12), suggestive of a biological role. Specifically, under physiological conditions, b-elimination effectively degrades D-serine, thus modulating the concentration of the neurotransmitter, especially in those areas of the brain lacking DAAO. It is

Table 1. Effect of effectors and post-translational modifications on SR activity

Effector/post translational modification	Species/tissue	Reaction	Effect	Reference
Mg-ATP	mSR	b-elimination (L-Ser)	k_cat/K_M (4x)	(34, 55)
	“	b-elimination (D-Ser)	k_cat/K_M (13x)	(55)
	“	Racemization (L-Ser)	k_cat/K_M (2x)	(34, 55)
	“	Racemization (D-Ser)	k_cat/K_M (7x)	(55)
S-nitrosylation of Cys113	mSR	Racemization (L-Ser)	Specific activity (¯2x)	(61)
			V_max (¯)
Ca⁺⁺	mSR	Racemization (L-Ser)	Specific activity (< 2x)	(20, 21)
PIP2	mSR	Racemization (L-Ser)	Specific activity (¯; IC₅₀= 13 mM)	(62)

V_max (¯)

Palmitoylation

rSR/brain

Racemization (L-Ser)

Specific activity (¯10x)

(63)

Phosphorylation

mSR (on Thr71)

Racemization (L-Ser)

Specific activity (2x)

(64)

GRIP1

mSR/astrocytes

D-serine release from cells ()

(66)

Racemization

(+ 65%)

PICK1

mSR/astrocytes

D-serine release from cells ()

(71)

DISC-1

msr and hSR

D-serine release from cells ()

(74)

also conceivable that pyruvate produced by the eliminase reaction could play a role in neuronal energy metabolism, in a similar fashion as the product of serine dehydratase (27). Nevertheless, it has been calculated that the rate of production of pyruvate by SR is less than 0.1 % of that displayed by glycolysis (56).