Site-directed mutagenesis hydrogen bonding

The family of serine proteases has been subjected to intensive studies of site-directed mutagenesis. These experiments provide unique information about the contributions of individual amino acids to kcat and KM. Some of the clearest conclusions have emerged from studies in subtilisin (Ref. 9), where the oxyanion intermediate is stabilized by t>e main-chain hydrogen bond of Ser 221 and an hydrogen bond from Asn 155 (Ref. 2). Replacement of Asn 155 (e.g., the Asn 155— Ala 155 described in Fig. 7.9) allows for a quantitative assessment of the effect of the protein dipoles on Ag. ... 

The importance of hydrogen bonds for the redox potential of the Rieske cluster has been demonstrated by site-directed mutagenesis of... 

Fig. 14. Plot of the g values g,g ) and of the average g value g vs rhombicity (UJ of (a) wild type (open symbol) and variant forms (closed symbols) of the Rieske protein in yeast bci complex where the residues Ser 183 and Tyr 185 forming hydrogen bonds into the cluster have been replaced by site-directed mutagenesis [Denke et al. (35) Merbitz-Zahradnik, T. Link, T. A., manuscript in preparation] and of (b) the Rieske cluster in membranes of Rhodobacter capsulatus in different redox states of the quinone pool and with inhibitors added [data from Ding et al. (79)]. The solid lines represent linear fits to the data points the dashed lines reproduce the fits to the g values of all Rieske and Rieske-type proteins shown in Fig. 13.

Figure 6.5 Topographical interaction model of features are red for positively ionizable, green for ccia adrenergic receptor generated on the basis of hydrogen bond acceptors, light blue for public site-directed mutagenesis. Both hydrophobicandorangeforringaromatic.Shape...

U. H. Mortensen, S. J. Remington, K. Breddam, Site-Directed Mutagenesis on (Serine) Carboxypeptidase Y. A Hydrogen Bond Network Stabilizes the Transition State by Interaction with the C-Terminal Carboxylate Group of the Substrate , Biochemistry 1994, 33, 508-513. 

The contributions of hydrogen bond donors to catalysis can be estimated by site-directed mutagenesis studies in cases where the hydrogen bond donor is located in the amino acid side chain. Deletion of the main chain NH is only possible by substituting the amino acid with a proUne. In all cases, the effects of the substitution to key enzyme kinetic parameters, and K, should be checked. Typically, the oxyanion hole residues contribute only Uttle to the binding of substrate [19-21]. This is reflected in the values, which typically remain very similar... 

The results of site-directed mutagenesis analysis of zinc ligands of higher plant p-carbonic anhydrase and of P. purpureum carbonic anhydrase have confirmed that zinc is essential for catalysis. X-ray fine structure data indicated that a water molecule is hydrogen bonded to the zinc-ligated Asp-151 and Asp-405. The water molecule is not directly coordinated to the zinc atom. A possible catalytic mechanism of C02 hydration cycle (211) has been proposed as given in Scheme 10. 

They also feature the same catalytic triad and the same system of hydrogen bonds for binding the carbonyl oxygen and the acetamido group of the substrate (Table 9.3). The bacterial enzymes are useful for site-directed mutagenesis studies because they are easily expressed in bacterial cells (for secretion) and because they do not require the activation reactions typical of the mammalian enzymes. 

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