Phosphoryl-aspartate

A sequence of ten amino acids (ICS-D-KTGTLT) around the phosphorylation site of Na,K-ATPase (Asp ) is highly conserved among the Na,K-, H,K-, Ca-, and Id-pumps [6]. There is also homology with the subunit of FpATP synthetase of mitochondria and chloroplasts (see [6]) except that Asp is replaced by Thr. Accordingly a covalent phosphorylated intermediate is not formed in Fi-ATPase. Mutagenesis of the phosphorylated aspartate residue in Na,K-ATPase [82], Ca-ATPase [87], or H-ATPase [88] completely blocks activity. 

Scheme 2 The catalytic mechanism of phosphorylation. Aspartate acts as a general base to deprotonate the hydroxyl group for nucleophilic attack at the 7-phosphate of ATP.

LT-aspartokinase <10> (<10> lysine-threonine-sensitive isoenzyme [3]) [3] aspartate kinase (phosphorylating) aspartate kinase III <11> [33] aspartic kinase... 

Acylphosphate formation is characteristic for P-type ATPases and involves the transfer of the y-phosphate of ATP to an aspartic acid residue to form a high-energy enzyme intermediate. The phosphorylated aspartic acid residue is located in the sequence DKTGT, which is universally conserved in all members of the P-type superfamily. By this criterion, CopA and CopB of En. hirae are clearly members of the P-type superfamily of ATPases and probably function by the same underlying mechanism. Vanadate sensitivity is another hallmark of P-type ATPases. CopA and CopB were inhibited by vanadate with /50 values of around 0.1 mM. This is a low vanadate sensitivity compared to /50 values in the micromolar to submicromolar range observed for non-heavy metal P-type ATPases. 

The synthesis of the other members of the aspartate family (Figure 14.8) is initiated by aspartate kinase (often referred to as aspartokinase) in an ATP-requiring reaction in which the side chain carboxyl group is phosphorylated. Aspartate... 

Aspartate kinase thrA metL lysC Phosphorylate aspartate to form aspartyl-P. There are three coding genes (thrA rnetL, lysC) in E. coli, but only one lysC) in C. glutamicum... 

Because the 2 NADH formed in glycolysis are transported by the glycerol phosphate shuttle in this case, they each yield only 1.5 ATP, as already described. On the other hand, if these 2 NADH take part in the malate-aspartate shuttle, each yields 2.5 ATP, giving a total (in this case) of 32 ATP formed per glucose oxidized. Most of the ATP—26 out of 30 or 28 out of 32—is produced by oxidative phosphorylation only 4 ATP molecules result from direct synthesis during glycolysis and the TCA cycle. 

Oxidation of 2 molecules each of isocitrate, n-ketoglutarate, and malate yields 6 NADH Oxidation of 2 molecules of succinate yields 2 [FADHg] Oxidative phosphorylation (mitochondria) 2 NADH from glycolysis yield 1.5 ATP each if NADH is oxidized by glycerol-phosphate shuttle 2.5 ATP by malate-aspartate shuttle + 3 + 5... 

Histidine phosphatases and aspartate phosphatases are well established in lower organisms, mainly in bacteria and in context with two-component-systems . Reversible phosphorylation of histidine residues in vertebrates is in its infancy. The first protein histidine phosphatase (PHP) from mammalian origin was identified just recently. The soluble 14 kD protein does not resemble any of the other phosphatases. ATP-citrate lyase and the (3-subunit of heterotrimeric GTP-binding proteins are substrates of PHP thus touching both, metabolic pathways and signal transduction [4]. 

Huang, Y. S., Jung, M. Y., Sarkissian, M., and Richter, J. D. (2002). N-methyl-D-aspartate receptor signaling results in Aurora kinase-catalyzed CPEB phosphorylation and alpha CaMKII mRNA polyadenylation at synapses. EMBOJ. 21, 2139-2148. 

This list is not intended to be comprehensive but to indicate the wide array of neuronal proteins regulated by phosphorylation. Some of the proteins are specific to neurons but most are present in many cell types in addition to neurons and are included because their multiple functions in the nervous system include the regulation of neuron-specific phenomena. Not included are the many phosphoproteins present in diverse tissues, including brain, that play a role in generalized cellular processes, such as intermediary metabolism, and that do not appear to play a role in neuron-specific phenomena. NMDA, N-methyl-D-aspartate CREB, cAMP response element-binding proteins STAT, signal-transducing activators of transcription ... 

Lau, L. F. and Huganir, R. L. Differential tyrosine phosphorylation of N-methyl-D-aspartate receptor subunits. /. Biol. Chem. 270 20036-20041,1995. 

Moon, I. S., Apperson, M. L. and Kennedy, M. B. The major tyrosine-phosphorylated protein in the postsynaptic density fraction is N-methyl-D-aspartate receptor subunit 2B. Proc. Natl Acad. Sci. U.S.A. 91 3954-3958,1994. 

Tezuka, T., Umemori, H., Akiyama, T. et al. PSD-95 promotes Fyn-mediated tyrosine phosphorylation of the N-methyl-D-aspartate receptor subunit NR2A. Proc. Natl Acad. Sci. U. S. A. 96 435-440,1999. 

Like aspartic peptidases, metallopeptidases act by activating a H20 molecule, and they do not form a covalent intermediate with the substrate. Here, the activation of a H20 molecule is mediated by a residue that acts as general base (e.g., Glu, His, Lys, Arg, or Tyr), with a divalent cation (usually Zn2+ but sometimes Co2+ or Mn2+) perhaps also contributing. The major role of the metal cation, however, is to act as an electrophilic catalyst by coordinating the carbonyl (or phosphoryl) O-atom in the substrate and orienting the latter for nucleophilic attack by the HO ion generated from H20 by the general base. 

The protein kinase mechanism uses aspartate as a general base, weakening the hydrogen—oxygen bond of the hydroxyl-containing residues. This allows the hydroxyl nucleophile of the substrate to attack the 7-phosphate of ATP, producing a phosphorylated peptide product and ADP (Scheme 2). 

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