Kinetic and equilibrium mechanisms of substrate binding to Mycobacterium tuberculosis enoyl reductase: implications to function-based antitubercular agent design

Abstract

A tuberculose (TB) continua sendo a principal causa de mortalidade devido a um único patógeno bacteriano, o Mycobacterium tuberculosis. Há, portanto, a necessidade de desenvolvimento de novos agentes antimicobacterianos. A 2-trans-enoil-ACP(CoA) redutase (InhA) de M. tuberculosis é o principal alvo da isoniazida. Aqui nós apresentamos dados de equilíbrio e cinética em estado pré-estacionário da ligação do substrato 2-trans-dodecenoil-CoA à InhA. Os resultados demonstram cooperatividade homotrópica positiva da ligação deste substrato à InhA e um processo de associação bimolecular seguido por uma lenta isomerização do complexo binário enzima-substrato. Os dados aqui descritos devem auxiliar no desenho racional de novos inibidores de um alvo protéico validado e com potencial utilização no tratamento da TB.Tuberculosis (TB) remains the leading cause of mortality due to a single bacterial pathogen, Mycobacterium tuberculosis. There is a need for the development of new antimycobacterial agents. M. tuberculosis 2-trans-enoyl-ACP(CoA) reductase (InhA) is the main target of isoniazid, the most prescribed anti-TB agent. Here we present pre-steady state kinetics and equilibrium data of 2-trans-dodecenoyl-CoA substrate binding to InhA. These results indicate both positive homotropic cooperativity upon substrate binding to InhA, and a bimolecular association process followed by a slow isomerization of the enzyme-substrate binary complex. The data here described should help the rational design of new agents against a validated and druggable protein target with potential anti-TB activity.

Observed crowding effects on Mycobacterium tuberculosis 2-trans-enoyl-ACP (CoA) reductase enzyme activity are not due to excluded volume only

“…Significant correlations between increasing concentrations of sucrose and decreasing k cat and K 0.5 values for DD-CoA were observed (Table ST2 ). As InhA follows a rapid-equilibrium random-order mechanism 9 , 10 , 14 , 36 , the decreasing k cat / K m values for NADH (Table ST1 ) in sucrose solution represents a solid piece of experimental evidence (even though not statistically significant). Moreover, the MD simulation results lend support to the effects of sucrose on InhA enzyme.…”

“…The specificity constant ( k cat / K m ) gives an estimate for the apparent second-order rate constant either for DD-CoA binding to InhA:NADH binary complex (at fixed-saturating NADH concentration and varying DD-CoA concentrations) or NADH binding to InhA:DD-CoA binary complex (at fixed-saturating DD-CoA concentration and varying NADH concentrations). As linear reciprocal plots were observed for both substrates of InhA 9 , 10 and NADH and DD-CoA have been shown to bind to free enzyme 14 , 36 , a rapid-equilibrium random-order mechanism appears to hold for InhA. Incidentally, it has been put forward that the K m for each substrate in this type of mechanism represents the equilibrium dissociation constant for dissociation of the substrate from the ternary complex 37 .…”

“…To ascertain that the values were fitted to the correct equation, the residual values were plotted versus DD-CoA concentration for ficoll 70 (Figure SF4 ), showing that a sigmoidal profile more appropriately describes the experimental results. It has been shown positive cooperativity ( n = 2) upon DD-CoA binding to free wild-type InhA, and a sequential or induced fit model with only one form of free InhA in solution was invoked to account for the results 36 . On the other hand, stopped-flow data on NADH binding to free isoniazid-resistant mutant InhA proteins suggested a mechanism with two forms of enzyme in equilibrium and only one of them being able to bind NADH 32 .…”

“…On the other hand, a sigmoidal kinetics without true cooperativity may be exhibited by a two-substrate enzyme that follows a random mechanism 17 . InhA is a two-substrate enzyme that follows rapid-equilibrium random-order mechanism, and a value of 0.57 μM ( K D ) for the hyperbolic binding of NADH to InhA 14 and a value of 8.2 μM ( K 0.5 ) for the sigmoidal binding of DD-CoA to InhA 36 were reported. The rate of InhA:DD-CoA → InhA:DD-CoA:NADH → products may be faster than InhA:NADH → InhA:NADH:DD-CoA → products reaction path for InhA.…”

“…Accordingly, polarity likely plays no role in sucrose solutions as no effects on steady-state kinetic parameters were observed for ficoll 70 and dextran 70 (Figs 1 and 2 ) as will be subsequently discussed. It has been reported that there occurs a quench in intrinsic protein fluorescence upon NADH 14 and DD-CoA 36 binding to free InhA. To evaluate whether the effects on steady-state kinetics parameters observed for sucrose were due to InhA:sucrose binary complex formation, intrinsic protein fluorescence measurements of sucrose binding to InhA were carried out.…”