PubMed · 11979874
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Tadashi Shimoyama, Shinsaku Fukuda, Qiang Liu, Kazuo Sugawara. 2002. [Rebamipide].. https://pubmed.ncbi.nlm.nih.gov/11979874/
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The development of renin inhibitors for the treatment of hypertension requires highly sensitive substrates to evaluate potency and to characterize the mechanism of tight-binding inhibitors. A series of intramolecularly quenched fluorogenic renin substrates, based on the N-terminal tetradecapeptide sequence of human angiotensinogen (hTDP), was synthesized using a solid-phase technique. Incorporation of the fluorescent amino acid L-Amp [L-2-amino-3-(7-methoxy-4-coumaryl)propionic acid] and the DNP (2,4-dinitrophenyl) group at various positions resulted in >90% quenching efficiency and strong product fluorescence. Shortening the hTDP sequence to an octapeptide from histidine in P5 to histidine in P3' (substrate 3) resulted in an acceptable k(cat)/K(m) (41000 M(-1).s(-1)) and further systematic variation gave substrate 9, DNP-Lys-His-Pro-Phe-His-Leu-Val-Ile-His-L-Amp, with a k(cat)/K(m) value of 350000 M(-1).s(-1) and 94% quenching efficiency. The free side chain of lysine, replacing the isoleucine residue at P6 position in the angiotensinogen sequence, contributed to the increased value for k(cat). The pH dependence of k(cat)/K(m) for renin and substrate 9 showed that the optimal pH is at pH 6-7. It also showed two titrating groups on the acidic side of the pH optimum, and one titrating group with a pK(a) of 7.8 on the alkaline side. The combination of good kinetic and spectroscopic properties resulted in a >20-fold improvement in the sensitivity of renin assay, compared with the commercial substrate Arg-Glu(EDANS)-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-Lys(DABCYL)-Arg [where EDANS is 5-[(2-aminoethyl)amino]naphthalene-1-sulphonic acid and DABCYL is 4-(4-dimethylaminophenylazo)benzoic acid] (k(cat)/K(m)=268000 M(-1) x s(-1), quenching efficiency <80%). The detection limit in a microplate renin assay was 60 pM, making substrate 9 well suited for the evaluation of inhibitors at picomolar concentrations.
The binding of enoyl-ACP (acyl-carrier protein) reductase from Plasmodium falciparum (PfENR) with its substrates and inhibitors has been analysed by SPR (surface plasmon resonance). The binding of the substrate analogue crotonoyl-CoA and coenzyme NADH to PfENR was monitored in real time by observing changes in response units. The binding constants determined for crotonoyl-CoA and NADH were 1.6x10(4) M(-1) and 1.9x10(4) M(-1) respectively. Triclosan, which has recently been demonstrated as a potent antimalarial agent, bound to the enzyme with a binding constant of 1.08x10(5) M(-1). However, there was a 300-fold increase in the binding constant in the presence of NAD+. The increase in the binding constant was due to a 17 times increase in the association rate constant (k(1)) from 741 M(-1) x s(-1) to 1.3x10(4) M(-1) x s(-1) and a 16 times decrease in the dissociation rate constant (k(-1)) from 6.84x10(-3) s(-1) to 4.2x10(-4) s(-1). These values are in agreement with those determined by steady-state kinetic analysis of the inhibition reaction [Kapoor, Reddy, Krishnasastry, N. Surolia and A. Surolia (2004) Biochem. J. 381, 719-724]. In SPR experiments, the binding of NAD+ to PfENR was not detected. However, a binding constant of 6.5x10(4) M(-1) was obtained in the presence of triclosan. Further support for these observations was provided by the crystal structures of the binary and ternary complexes of PfENR. Thus the dramatic enhancement in the binding affinity of both triclosan and NAD+ in the ternary complex can be explained by increased van der Waals contacts in the ternary complex, facilitated by the movement of residues 318-324 of the substrate-binding loop and the nicotinamide ring of NAD+. Interestingly, the results of the present study also provide a rationale for the increased affinity of NAD+ for the enzyme in the ternary complex.
13C-NMR has been used to follow the tryptophan synthase (EC 4.2.1.20) catalysed hydrogen-deuterium exchange of the pro-2R and pro-2S protons of [2-13C]glycine at pH 7.8. 1H-NMR has also been used to follow the tryptophan-synthase-catalysed hydrogen-deuterium exchange of the alpha-protons of a range of L- and D-amino acids at pH 7.8. The pK(a) values of the alpha-protons of these amino acids have been estimated and we have determined whether or not their exchange rates can be predicted from their pK(a) values. With the exception of tryptophan and norleucine, the stereospecificities of the first-order alpha-proton exchange rates are independent of the size and electronegativity of the amino acid R-group. Similar results are obtained with the second-order alpha-proton exchange rates, except that both L-tryptophan and L-serine have much higher stereospecificities than all the other amino acids studied.