Pseudosubstrate

Cyclic AMP-dependent protein kinase is shown complexed with a pseudosubstrate peptide (red). This complex also includes ATP (yellow) and two Mn ions (violet) bound at the active site. 

Protein Kinase C. Figure 1 Domain structure of PKC family members showing regulatory modules (pseudosubstrate sequence and C1, C2, and PB1 domains) and the kinase core. Shown below are the structures of the C1 domain of PKC 5 with bound phorbol (purple), the C2 domain of PKC (3 with bound Ca2+ (pink spheres), and the recently solved structure of the kinase domain by Grant and coworkers [1] of PKC pil with phosphorylation sites indicated in pink. Figure adapted from Newton (2003). 

The term pseudosubstrate as used in this article will comprise sugar-related compounds that are chemically transformed by glycosidases, often forming long-lived intermediates and thereby acting as reversible inhibitors. Even in cases of weak inhibition, where the intermediate is too short-lived for chemical or physical characterization, the type of reaction catalyzed by the... 

A transition to irreversible inhibitors is seen in pseudosubstrates forming enzyme-bound intermediates which are cleaved very slowly, for example, the 2-deoxy-2-fluoro-D-glycosides to be discussed in Section II,3,b. 

Some nonreactive molecules are recognized by the target enzyme as pseudosubstrates. These bind to the enzyme active site and are chemically transformed into reactive species that then covalently inactivate the enzyme. 

All isoforms of PKC are predominantly localized to the cytosol and, upon activation, undergo translocation to either plasma or nuclear membranes. However, newly synthesized PKCs are localized to the plasmalemma and are in an open conformation in which the auto inhibitory pseudosubstrate sequence is removed from the substrate binding domain. The maturation of PKC isoforms is effected by phosphoinositide-dependentkinase-I (PDK-I), which phosphorylates a conserved threonine residue in the activation loop of the catalytic (C4) domain [24]. This in turn permits the autophosphorylation of C-terminus threonine and serine residues in PKC, a step which is a prerequisite for catalytic activity (see also Chs 22 and 23). The phosphorylated enzyme is then released into the cytosol, where it is maintained in an inactive conformation by the bound pseudosubstrate. It was originally thought that 3-phosphoinositides such as PI(3,4)P2 and PI(3,4,5)P3 could directly activate PKCs. However, it now seems more likely that these lipids serve to activate PDK-1 (a frequent contaminant of PKC preparations). 

Scheme 5. Oxidation of substrates and pseudosubstrates catalyzed by copper oxidases.

The mechanism of the aspartic proteinases involves two essential catalytic aspartate residues. There is some controversy in the literature as to whether the mechanism involves an acyl en me intermediate or an amino enzyme intermediate (4). However, there is no direct evidence for either intermediate so additional studies with inhibitors and pseudosubstrates along with crystallographic analysis will ultimately be required to resolve these questions. 

Protein Kinase Inhibitors Pseudosubstrate-based peptide inhibitors, 201, 287 utilization of the inhibitor protein of adenosine cyclic monophosphate-dependent protein kinase, and peptides derived from it, as tools to study adenosine cyclic monophosphate-mediated cellular processes, 201, 304 use of sphingosine as inhibitor of protein kinase C, 201, 316 properties and use of H-series compounds as protein kinase inhibitors, 201, 328 use and specificity of staurosporine, UCN-01, and calphostin C as protein kinase inhibitors, 201, 340 inhibition of protein-tyrosine kinases by tyrphostins, 201, 347 use and specificity of genistein as inhibitor of protein-tyrosine kinases, 201, 362 use and selectivity of herbimycin a as inhibitor of protein-tyrosine kinases,... 

Carboxypeptidase A was the first zinc enzyme to yield a three-dimensional structure to the X-ray crystallographic method, and the structure of an enzyme-pseudosubstrate complex provided a model for a precatalytic zinc-carbonyl interaction (Lipscomb etai, 1968). Comparative studies have been performed between carboxypeptidase A and thermolysin based on the results of X-ray crystallographic experiments (Argosetai, 1978 Kesterand Matthews, 1977 Monzingoand Matthews, 1984 Matthews, 1988 Christianson and Lipscomb, 1988b). Models of peptide-metal interaction have recently been utilized in studies of metal ion participation in hydrolysis (see e.g., Schepartz and Breslow, 1987). In these examples a dipole-ion interaction is achieved by virtue of a chelate interaction involving the labile carbonyl and some other Lewis base (e.g.. 

We have approached this problem by studying the interactions between pepsin and ketones with structures based on that of pepstatin. Our strategy was to design ketones which would serve as pseudosubstrates, that is, be subject to the catalytic action of the enzyme, but only to the point of formation of a tetrahedral intermediate which, because of the increased stability of a C-C vs a C-N bond, would not break down to products. Such a stable tetrahedral intermediate would then, in principle, be amenable to study by the appropriate physical methods. appeared to be an ideal method since changes... 

Figure 6. Schematic representation of the addition of water to the labeled carbonyl in statone pseudosubstrate 6. Labeling of water 2H2 0 or 2h21 0) establishes that water not Asp-32 adds to the carbonyl group (Cf text).

Pseudosubstrates in which the fluorinated group is introduced to destabilize a reaction intermediate or a transition state of the enzymatic transformation. This can provoke inhibition or irreversible inactivation of the enzyme. 

Ser residue to be phosphorylated this is replaced by alanine. Due to these characteristics, the inhibitor peptide is bound in a very similar way and with similar affinity to a substrate, but it carmot react and has the property of a pseudosubstrate. The contacts between protein kinase A and a substrate are shown as a model for a peptide known as kemptide, which serves as a phosphorylation substrate. 

If the inhibitory structural element is itself a part of the protein kinase, this is known as autoinhibition. The inhibitory structural elements often have the character of a pseudosubstrate. They possess a similarity to the proper substrate and can accumulate in the substrate binding site. Since the pseudosubstrate lacks a phosphate receptor, no phosphorylation takes place (review Kemp et al., 1994). 

Forming contacts with the substrate binding site (pseudosubstrate function)... 

The members of the protein kinase C family are composed of a polypeptide chain with a molecular weight of 68-83 kDa. The N-terminal regulatory domains Cl and C2 and a C-terminal catalytic domain can de differentiated in the primary structure (Fig. 7.8). In addition, a pseudosubstrate sequence with autoinhibitory function is located at the N-terminus. 

Fig. 7.8. Functional domains of protein kinase C. The functional domains of protein kinase Ca and C8 are shown as a linear representation. The binding site for TPA lies in domain Cl. Domain C2 contains the Ca binding site. Protein kinase C8 lacks the C2 elements and thus regulation by Ca. According to Azzi et ah, (1992). Pseudosubstrate autoinhibitory sequence with pseudosubstrate character.

In the absence of activating cofactors, the catalytic domain is subject to autoinhibition by the regulatory domain (Orr and Newton, 1994). A sequence motif is found in the regulatory domain which serves as a pseudosubstrate. It resembles the consensus sequence for phosphorylation sites of protein kinase C but does not have a Ser or Thr residue for phosphorylation. This sequence motif is found in aU protein kinase C family members. It is assumed that the active center is inhibited by occupation by the pseudosubstrate. 

Kemp, B.E., Parker, M.W., Hu, S., Tiganis, T. and House, C. Substrate and pseudosubstrate interactions with protein kinases determinants of specificity (1994) Trends Bioch. Sci. 19, 441-448... 

In contrast, selective inhibition of enzyme activity involves highly specific interactions between the protein and chemical groups on the xenobiotic. An excellent example of this type of inhibition is seen in the toxic effect of fluoroacetate, which is used as a rodenticide. Although fluoroacetate is not directly toxic, it is metabolized to fluoroacetyl-CoA, which enters the citric acid cycle due to its structural similarity to acetyl-CoA (Scheme 3.5). Within the cycle, fluoroacetyl-CoA combines with oxalo-acetate to form fluorocitrate, which inhibits the next enzyme, aconitase, in the cycle [42]. The enzyme is unable to catalyze the dehydration to cis-aconitate, as a consequence of the stronger C-F bond compared with the C-H bond. Therefore, fluorocitrate acts as a pseudosubstrate, which blocks the citric acid cycle and, subsequently, impairs ATP synthesis. 

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