Enzymes as targets for pharmaceutical intervention

In the next peiragraph, we describe our work on the meiin targets for therapeutic intervention in AIDS, the enzymes protease and reverse transcriptase from human immunodeficiency virus type 1 (HIV-1 PR and HIV-1 RT). Subsequently, we focus on an 

HIV-1 PR cleaves the multidomain protein encoded by the virus genome to yield separated structural proteins. Structure-based drug-design studies have shown that in the substrate-cleavage site - two Asp-Thr-Gly loops at the subunit-subunit interface (Fig. 5a) - the almost coplanar conformation of the catalytic Asp dyad is crucial for enzymatic function and for the binding of both substrate and inhibitors [88-90]. 

Quantum-mechanical approaches appear ideally suited to provide an understanding of the underlying molecular intercictions of the Asp dyad. Here, we present results from our ab initio MD simulations [100]. This investigation, which focuses on the free enzyme, is divided in two steps. First, we attempt to determine the protonation state of the Asp 

Protonation State. At optimal pH for enzymatic activity ( 5-6) [101, 102, 105], the Asp dyad can in principle exist in three protonation states, a deprotonated, a mono-protonated or a doubly protonated form. Because hydrogen atoms cffe invisible in the X-ray structure, evidence for a specific protonation state must be inferred indirectly by spectroscopic or titration measurements. Up to now, the existence of the doubly protonated, neutral form h is not been proposed for the free enzyme. The existence of the deprotonated, doubly negative form is supported by a recent NMR study [102] at pH 6. However, this study has been subjected to criticism [106] and it is not conclusive. Our ab initio simulations of this form show that the Asp dyad is unstable even in the ps timesccde because of the strong Asp-Asp repulsion, which turns out to be -t-30 kcal/mol as estimated with a simple electrostatic model [100]). Thus, our calculations do not support the existence of this form. 

The third possible state is the mono-protonated one, which has been strongly suggested from both experiment and theory [101, 106]. 

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