Subtilisin structural relationships

A diagram illustrating the spatial relationships among His-64 and residues 99 and 156 of subtilisin is shown in Fig. 17. Protein structure intervenes between His-64 and position 99 (—13 A), and solvent intervenes between His-64 and position 156 (—15 A). Wild-type subtilisin has Asp-99 and Glu-156, and when either of these side chains is mutated to serine, the pAa of His-64 decreases by 0.4 (assayed at ionic strength 0.001 M). If both side chains are mutated to serines, the pAa of His-64 in the double-mutant decreases by 0.65. Hence, decreasing the amount of negative surface charge results in the expected pAa decrease for His-64. 

As opposed to the catalytic triad, which is made up of side chains that can now be mutated at will, the structure-function relationships in the oxyanion hole are not equally susceptible to experimental verification. Only in subtilisin the involvement of the side-chain amide of Asn-155 allows for quantitadve assessment of the role of the oxyanion H bonds in the stabilization of the tetrahedral intermediates. The replacement of Asn-155 with an isosteric Leu reduces the Acat by 200- to 300-fold, but leaves the Km essentially unaffected (Bryan et al., 1986). This observation is fully consistent with Asn-155 not contributing to substrate binding, but playing a key role in the stabilization of the intermediate. 

The earliest observation that implied evolutionary links between all lipases was that of the consensus pentapeptide G-X-S-X-G, subsequently shown to contain the nucleophilic serine. The apparent similarity of this sequence to that found around the active serine in the chy-motrypsin and subtilisin families of serine proteinases prompted a number of authors to infer an evolutionary relationship between the three families. Further evidence in support of such a link came from secondary structure prediction studies indicating that the nucleophilic serine in a lipase is most likely within a /3 turn, structurally reminiscent of proteinases (Reddy et ai, 1986). In fact, one of the commonly used phrases found in introductions to many papers dealing directly or indirectly with lipases refers to the consensus G-X-S-X-G pentapeptide found in the active site of all serine proteinases and esterases. We now know that the implication that homology and/or structural similarities exist between the enzymes belonging to these diverse groups is incorrect. The matter has been dealt with in the literature (Derewenda and Derewenda, 1991 Liao et ai, 1992), but it seems appropriate to review some of the conclusions. 

Subtilisin, a bacterial proteolytic enzyme originally isolated from Bacillt4S subtilus, is a serine protease. Even though its primary and tertiary structures bear no discernible relationship to chymotrypsin, the active site groups and the... 

STRUCTURE OF THERMITASE, A THERMOSTABLE SERINE PROTEINASE FROM THERMOACTINOMYCES VULGARIS, AND ITS RELATIONSHIP WITH SUBTILISIN-TYPE PROTEINASES... 

Corresponding equations were derived for the cysteine proteases ficin, actinidin, bromelain B and D, and the serine proteases subtilisin, chymotrypsin, and trypsin. In all cases, the coefficient of the electronic a term is around 0.4-0.7. Whereas the other regression coefficients in equations (16) and (17) are also relatively similar, the constant terms of both equations differ by about 1.8 log units, which indicates that the lower lipophilicity of the mesyl group, as compared with a much more lipophilic benzoyl group, is responsible for the lower binding affinities of the mesyl amides. To prove this hypothesis, a series of (4-X-Phe)-CONHCH2COO-pyrid-3-yl analogs 12 was synthesized and tested. As expected, lipophilicity determines the structure-activity relationship in this part of the molecules (equation IS). " " ... 

A great similarity of part of structure of subtilisin and lactate dehydrogenase has been noted (Rao and Rossmaim, 1973). In subtilisin and in carboxypeptidase four parallel central strands of p pleated sheets are super-posable (Rossmann and Argos, 1977). Since these similar structural patterns form structural domains in the corresponding proteins, their evolutionary relationship is discussed in the following paragraphs. 

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