Kinase definition

Multiple physiological roles for IP6 and the diphosphoinositol polyphosphates have been proposed, including effects on endocytosis and mRNA transport [3] however, definitive evidence for many of these functions is lacking. Such studies are complicated by possible nonspecific effects of this highly negatively charged molecule. Of note is the report of an IP6/IP7-dependent protein kinase activity that phosphorylates pacsin/syndapin I, a protein involved in synaptic vesicle recycling [21]. 

The catalytic center is formed by residues from both lobes. Sequence comparisons, mutation experiments and biochemical studies indicate an essential fimction in catalysis of phosphate transfer for the conserved amino acids Lys72, Aspl66 and Aspl84 (numbering of PKA). However, the catalytic mechanism of phosphate transfer is not definitely established. It is generally assumed that Aspl66, which is invariant in all protein kinases, serves as a catalytic base for activation of the Ser/Thr hydroxyl and that the reaction takes place by an in-line attack of the Ser-OH at the y-phosphate. 

In fact, kinetic studies of the GTP-dependent avian mitochondrial enzyme indicate two metal-binding sites, one on the polyphosphate group of the bound GTP and one on carboxylate side chains of the protein.252 255 The three-dimensional structure of the ATP-dependent E. coli enzyme reveals a nucleotide binding site similar to the ATP site of adenylate kinase (Fig. 12-30).256 A definite binding site for C02 is also present in the enzyme.257... 

The separateness of two domains within a subunit varies all the way from independent globular domains joined only by a flexible length of polypeptide chain, to domains with tight and extensive contact and a smooth globular surface for the outside of the entire subunit, as in the proteolytic enzyme elastase (fig. 4.20). An intermediate level of domain separateness, characterized by a definite neck or cleft between the domains, is found in phosphoglycerate kinase (fig. 4.21). 

The present chapter focuses on the physiological functions of the CaMK cascade that have been identified to date. The definition of these functions has relied heavily on the use of pharmacological inhibitors, overexpression of constitutively active or dominant/negative mutants of the various CaM kinases, and a variey of model organisms that include mice, worms and fungi null (or transgenic) for specific members of the CaMK cascade. 

A possible dual action of growth factors on kinases and on the oxidase makes definition of separate effects difficult. Further complication arises with cytokines which induce H202 production by receptive cells, since H202 itself may activate proton movement and cell proliferation (Meier et al., 1990 Shibanuma et al., 1990 Kaufman et al., 1993 Yuo et al., 1993). 

The multienzymic nature of pyruvate kinase has been investigated in detail in M. expansa (98), H. diminuta (117) and S. erinacei (240). These worms possess FBP-sensitive and FBP-insensitive pyruvate kinase isoenzymes. In H. diminuta, as many as five pyruvate kinase isoenzymes (for definition and usefulness, see Chapter 6) occur during development (Fig. 5.3) and it seems likely that differential expression of these different forms of the enzyme may help to control the specific composition of excreted end-products by the various life cycle stages. The nature and regulation of the end-products secreted in H. diminuta are discussed further below. 

Therefore, the availability of purified enzymes specific to PolyPs allowed the development of rapid, sensitive and definitive assays. It should be noted, however, that PolyPs in biological samples may not be effectively hydrolysed by exopolyphosphatase or be available for polyphosphate kinase (Sethuraman et al, 2001). 

Rapid phosphorylation of the other detected phosphoproteins does occur but no definite roles have yet been ascribed to them. The 33 kDa protein may be the S6 ribosomal protein involved in the control of protein synthesis. The 57 kDa protein has been identified as the regulatory suhunit of the cyclic AMP-dependent protein kinase [44]. Of the other proteins the 76, 43 and 20 kDa may be connected with the microfilaments (43 kDa actin, 76 kDa myosin light chain kinase and 20 kDa myosin light chain) but this must be further investigated. These proteins may only play a permissive role in. steroidogenesis. The fact that the pattern of protein phosphorylation is very similar after stimulation of protein kinase C with phorbol esters supports this because the latter only marginally increase steroidogenesis [18]. 

---