Creatine substrate binding

Dodd, J. R. and Christie, D. L. (2001) Cysteine 144 in the third transmembrane domain of the creatine transporter is located close to a substrate-binding site.. /. Biol. Chem. 276, 46983-46988. 

The overall direction of the reaction will be determined by the relative concentrations of ATP, ADP, Cr, and CrP and the equilibrium constant for the reaction. The enzyme can be considered to have two sites for substrate (or product) binding an adenine nucleotide site, where ATP or ADP binds, and a creatine site, where Cr or CrP is bound. In such a mechanism, ATP and ADP compete for binding at their unique site, while Cr and CrP compete at the specific Cr-, CrP-binding site. Note that no modified enzyme form (E ), such as an E-PO4 intermediate, appears here. The reaction is characterized by rapid and reversible binary ES complex formation, followed by addition of the remaining substrate, and the rate-determining reaction taking place within the ternary complex. 

The function of magnesium in enzyme activity may either be to form a complex with the substrate, as in the magnesium-ATP complex formed in creatine kinase and phosphofhictokinase, or to bind to the enzyme and either produce an allosteric activation or play a direct role in catalysis. If an enzyme is known to utilize a nucleotide as one of its substrates, it can be assumed that magnesium is also required for catalysis. The magnesium ion possibly acts as an electrostatic shield. The enzyme pyravate kinase, described earlier, and shown in Figure 1, requires both magnesium and potassium ions for maximal activity. 

The DAT, NET, and SERT are members of the family of Na+, Cl -dependent substrate-specific neuronal membrane transporters, which includes transporters for GABA, glycine, taurine, proline, betaine, and creatine (4-8). The putative structure of these transporters consists of 12 transmembrane domains with both the N- and C-terminal domains located within the cytoplasm. The mechanism of the transporter-mediated uptake of monoamines is believed to involve an electrogenic transport of monoamines by sequential binding and cotransport of Na+ and Cl-ions (4-8). 

Equation 2.28 contains a new term, Km 2, that represents a change in affinity of the enzyme for one substrate once the other substrate is bound. If the mechanism is ordered, the simple relationship Km 2 = Km x Km2 may be applied. For a random mechanism, the value of Km 2 is determined experimentally. Creatine kinase (CK) is an example of this type of enzyme. Creatinine and ATP bind to the enzyme randomly in nearby, but independent binding sites. 

A recent report (129) on the utilization of the (R f) and (5),p) isomers of ADP(a-S) (a thio substituted ADP) by Mn substituted creatine kinase has shown that the enzyme binds the (/ )(p) substrate with the Mn bound to the O but, with the (5)(p) substrate, the enzyme bound form is predominantly coordinated via the sulfur atom. Both these complexes result in the... 

Cerium forms complexes with riboflavin (Sek-HON and Chopra 1974). Cerium chloride (2 mg/kg rat intravenously) impaired the activities of cytochrome P-450 and NADPH-cytochrome c reductase of rat liver, which could be prevented by a-tocopherol acetate (100 mg/kgxday i.p.) pre-treatment (Ar-VELA 1974). Ce promoted the binding of creatine kinase to Cibacron blue F3 GA, the substrate analogue of the enzyme, even in the absence of Mg its native cofactor (Shivakumar et al. 1989). 

Metal cofactors do not always bind to the enzyme but rather bind to the primary substrate. The resulting substrate-metal complex binds to the enzyme and facilitates its activity. Creatine kinase catalyses the transfer of phosphoryl groups from adenosine triphosphate (ATP), which is broken down to adenosine diphosphate (ADP). The reaction requires the presence of magnesium ions. These, however, do not bind to the enzyme but bind to ATP, forming an ATP Mg complex. It is this complex that binds to the enzyme and allows transfer of the phosphoryl group ... 

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