M. A. Qasim

What can the structures of enzyme-inhibitor complexes tell us about the structures of enzyme substrate complexes?

Proteinases perform many beneficial functions that are essential to life, but they are also dangerous and must be controlled. Here we focus on one of the control mechanisms: the ubiquitous presence of protein proteinase inhibitors. We deal only with a subset of these: the standard mechanism, canonical protein inhibitors of serine proteinases. Each of the inhibitory domains of such inhibitors has one reactive site peptide bond, which serves all the cognate enzymes as a substrate. The reactive site peptide bond is in a combining loop which has an identical conformation in all inhibitors and in all enzyme-inhibitor complexes. There are at least 18 families of such inhibitors. They all share the conformation of the combining loops but each has its own global three-dimensional structure. Many three-dimensional structures of enzyme-inhibitor complexes were determined. They are frequently used to predict the conformation of substrates in very short-lived enzyme-substrate transition state complexes. Turkey ovomucoid third domain and eglin c have a Leu residue at P(1). In complexes with chymotrypsin, these P(1) Leu residues assume the same conformation. The relative free energies of binding of P(1) Leu (relative to either P(1) Gly or P(1) Ala) are within experimental error, the same for complexes of turkey ovomucoid third domain, eglin c, P(1) Leu variant of bovine pancreatic trypsin inhibitor and of a substrate with chymotrypsin. Therefore, the P(1) Leu conformation in transition state complexes is predictable. In contrast, the conformation of P(1) Lys(+) is strikingly different in the complexes of Lys(18) turkey ovomucoid third domain and of bovine pancreatic trypsin inhibitor with chymotrypsin. The relative free energies of binding are also quite different. Yet, the relative free energies of binding are nearly identical for Lys(+) in turkey ovomucoid third domain and in a substrate, thus allowing us to know the structure of the latter. Similar reasoning is applied to a few other systems.

Additivity-based prediction of equilibrium constants for some protein–protein associations

For many protein families, such as serine proteinases or serine proteinase inhibitors, the family assignment predicts reactivity only in general terms. Both detailed specificity and quantitative reactivity are lacking. We believe that, for many such protein families, algorithms can be devised by defining the subset of n functionally important sequence positions, making the 19n possible single mutants and measuring their reactivity. Given the assumption that the contributions of the n positions are additive, the reactivities of the 20(n) variants can be predicted. This is illustrated by an almost complete algorithm for the Kazal family of protein inhibitors of serine proteinases.

Structure of the subtilisin Carlsberg-OMTKY3 complex reveals two different ovomucoid conformations.

One of the most studied protein proteinase inhibitors is the turkey ovomucoid third domain, OMTKY3. This inhibitor contains a reactive-site loop (Lys13I-Arg21I) that binds in a nearly identical manner to all studied serine proteinases, regardless of their clan or specificity. The crystal structure of OMTKY3 bound to subtilisin Carlsberg (CARL) has been determined. There are two complete copies of the complexes in the crystallographic asymmetric unit. Whereas the two enzyme molecules are virtually identical [0.16 A root-mean-square difference (r.m.s.d.) for 274 C(alpha) atoms], the two inhibitor molecules show dramatic differences between one another (r.m.s.d. = 2.4 A for 50 C(alpha) atoms). When compared with other proteinase-bound OMTKY3 molecules, these inhibitors show even larger differences. This work facilitates a re-evaluation of the importance of certain ovomucoid residues in proteinase binding and explains why additivity and sequence-based binding-prediction methods fail for the CARL-OMTKY3 complex.

MOLECULAR BASIS OF INDOMETHACIN-HUMAN SERUM ALBUMIN INTERACTION

Studies on the strength and extent of binding of the non‐steroidal anti‐inflammatory drug indomethacin to human serum albumin (HSA) have provided conflicting results. In the present work, the serum‐binding of indomethacin was studied in 55 mM sodium phosphate buffer (pH 7·0) at 28°C, by using a fluorescence quench titration technique.

Functional evolution within a protein superfamily

The ability to predict and characterize distributions of reactivities over families and even superfamilies of proteins opens the door to an array of analyses regarding functional evolution. In this article, insights into functional evolution in the Kazal inhibitor superfamily are gained by analyzing and comparing predicted association free energy distributions against six serine proteinases, over a number of groups of inhibitors: all possible Kazal inhibitors, natural avian ovomucoid first and third domains, and sets of Kazal inhibitors with statistically weighted combinations of residues. The results indicate that, despite the great hypervariability of residues in the 10 proteinase‐binding positions, avian ovomucoid third domains evolved to inhibit enzymes similar to the six enzymes selected, whereas the orthologous first domains are not inhibitors of these enzymes on purpose. Hypervariability arises because of similarity in energetic contribution from multiple residue types; conservation is in terms of functionality, with “good” residues, which make positive or less deleterious contributions to the binding, selected more frequently, and yielding overall the same distributional characteristics. Further analysis of the distributions indicates that while nature did optimize inhibitor strength, the objective may not have been the strongest possible inhibitor against one enzyme but rather an inhibitor that is relatively strong against a number of enzymes. Proteins 2006.

Investigation of the Mechanism of Protein Denaturation by Guanidine Hydrochloride-Induced Dissociation of Inhibitor-Protease Complexes

In this communication we describe an approach in which guanidine hydrochloride-induced dissociation of a protein inhibitor-serine protease complex is used to explore the molecular basis of protein denaturation. The rationale behind this approach is that the inhibitor-protease complex is stabilized by the same types of non-covalent interactions that stabilize the native state of a protein. The dissociation of inhibitor-protease complex can be performed at concentrations of guanidine hydrochloride at which the inhibitor and the protease retain their native conformations. Here, we present our results on the effect of 0.1 M to 0.4 M guanidine hydrochloride concentrations on the association equilibrium constants (reciprocal of dissociation constant) of P₁G, P₁A, P₁V, P₁N, and P₁S variants of turkey ovomucoid third domain with bovine α-chymotrypsin. We use these results to calculate the free energy change in the dissociation of inhibitor-protease complexes (the m value) per mol of guanidine hydrochloride concentration. Our results agree with the general consensus that the denaturing effect of guanidine hydrochloride is due to its favorable interaction with the polar parts of proteins and that the non-polar side chains have no or little favorable interaction with guanidine hydrochloride.

Additivity-based design of the strongest possible turkey ovomucoid third domain inhibitors for porcine pancreatic elastase (PPE) and Streptomyces griseus protease B (SGPB)

We describe here successful designs of strong inhibitors for porcine pancreatic elastase (PPE) and Streptomyces griseus protease B (SGPB). For each enzyme two inhibitor variants were designed. In one, the reactive site residue (position 18) was retained and the best residues were substituted at contact positions 13, 14, and 15. In the other variant the best residues were substituted at all contact positions except the reactive site where a Gly was substituted. The four designed variants were: for PPE, T13E14Y15‐OMTKY3 and T13E14Y15G18M21P32V36‐OMTKY3, and for SGPB, S13D14Y15‐OMTKY3 and S13D14Y15G18I19K21‐OMTKY3. The free energies of association (ΔG 0) of expressed variants have been measured with the proteases for which they were designed as well as with five other serine proteases and the results are discussed.