Asp dyad

Figure 2. A ribbon diagram of rhizopus pepsin (PDB code 5APR). The catalytically important Asp dyad (Asp218 and Asp35) side-chains are shown in stick diagrams. The P-hair pin flap that covers the active site cleft is located in the bottom of the diagram.

Like MR, NAAAR represents another racemase activity in the enolase superfamily. As would be expected, this enzyme contains machinery for initiation of its chemical reaction by abstraction of the a-proton from either an R- or an S-substrate. Thus, NAAAR possesses the S-specific base motif, KXK, as well an R-specific base, this time a Lys, in contrast to the His/Asp dyad in MR (Fig. 8). For this reason, NAAAR was originally assigned to the MLE subgroup, the structurally characterized member of the superfamily to which it is most similar (29% identical to the MLE I of P. putida) and which also has a Lys residue in the R-specific base position. Unlike any other members of the superfamily, however, NAAAR is a remarkably inefficient enzyme (Ka/Km = 3.7 X 102 M-1 s 1). 

The first theozyme (Scheme 9.4a) invokes a proton shuttle using lysine and aspartate. The second theozyme (Scheme 9.4b) has a tyrosine group to accept the proton from the alcohol, whereas the third theozyme (Scheme 9.4c) uses the His-Asp dyad to accept this proton. In the last theozyme (Scheme 9.4d), an explicit water molecule assists in die proton shuttling. 

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 hfree 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. 

Figure 7 HTV-l PR Thr26-Gly27 peptide unit s dipole (calculated and experimental (Nelson RD, et al. Nat l. Bur. Stands. 10, 1967) values 3.82 D and 3.84 D, respectively) superimposed on the electrostatic potential of the Asp dyad active site. The coloring varies continuously from red in negative areas to blue in more positive regions.(Reproduced with permission from ref. [100], Copyright 2000 Wiley.)...

Fig. 19. (a) Location of the proton between the Asp dyad (b, c, d) position of the proton as a function of time for models C(I), C(II), and C, respectively. Note that in d, only the last 0.9 ps is shown. Reprinted with permission from Carloni and Rothlisberger (2001). 

Pelmenschikov, V. Siegbahn, P.E.M. (2002). Catalytic Mechanism of Matrix Metalloproteinases Two-Layered Piana, S. Carloni, P. (2000). Conformational flexibility of the catalytic asp dyad in HIV-1 protease an ab initio study on the free enzyme. Proteins Struct, Funct. Genet, Vol. 39, No.l, pp. 26-36 Polgar, L. (1989). Mechanisms of Protease Action, pp 157-182, CRC Press, Inc., Boca Raton, FL Pollack, L. (2011a). SAXS Studies of Ion-Nucleic Acid InteractionsAnnual Review of Biophysics Vol 40,(June 2011),pp 225-242... 

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