Understanding the Functional Roles of Amino Acid Residues in Enzyme Catalysis

Although the cation-pi pair, formed between a side chain or substrate cation and the negative electrostatic potential of a pi system on the face of an aromatic ring, has been widely discussed and has been shown to be important in protein structure and protein-ligand interactions, there has been little discussion of the potential structural and functional importance in proteins of the related anion-aromatic pair (i.e., interaction of a negatively charged group with the positive electrostatic potential on the ring edge of an aromatic group). We posited, based on prior structural information, that anion-aromatic interactions between the anionic Asp general base and Phe54 and Phe116 might be used instead of a hydrogen-bond network to position the general base in the active site of ketosteroid isomerase from Comamonas testosteroni as there are no neighboring hydrogenbonding groups. We have tested the role of the Phe residues using site-directed mutagenesis, double-mutant cycles, and high-resolution X-ray crystallography. These results indicate a catalytic role of these Phe residues. Extensive analysis of the Protein Data Bank provides strong support for a catalytic role of these and other Phe residues in providing anion-aromatic interactions that position anionic general bases within enzyme active sites. Our results further reveal a potential selective advantage of Phe in certain situations, relative to more traditional hydrogen-bonding groups, because it can simultaneously aid in the binding of hydrophobic substrates and positioning of a neighboring general base.enzyme catalysis | general-base catalysis | noncovalent interactions E nzymes use the same functional groups to achieve "chemical catalysis" as small-molecule catalysts, and yet enzymes attain much greater rate enhancements. A distinguishing feature of enzymes is that their reactions occur in highly specialized active sites that use noncovalent interactions to precisely position enzymatic functional groups relative to substrates and relative to other active-site features. Understanding how these groups are positioned within enzyme active sites is key for understanding the differences between enzymes and small-molecule catalysts.It is generally recognized that hydrophobic interactions can help bind and position substrates in enzyme active sites (1, 2). Beyond hydrophobic interactions, much attention has focused on the importance of hydrogen bonds in precisely positioning active-site groups directly involved in the chemical reaction (e.g., the catalytic triad in serine proteases), and the importance of hydrogen-bonding groups is supported by mutagenesis in many cases (e.g., refs. 1 and 3-5).In addition to hydrogen bonds, the cation-pi pair, formed between a side-chain or substrate cation and the negative electrostatic potential associated with the face of a pi system, has been widely discussed in hundreds of literature reports and has been shown to be important in both protein structure and proteinligand interactions (e.g., refs. 6 and 7). There has been much ...

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confidence: 61%

Use of anion–aromatic interactions to position the general base in the ketosteroid isomerase active site

Schwans¹,

Sunden²,

Lassila³

et al. 2013

“…This study considers neither the mechanisms nor any cofactors used in catalysis (21), which has some advantages, because mechanistic information is difficult to validate experimentally and is therefore disputed in the literature. In addition, mechanistic components are usually not captured in reaction files, and the EC classification does not use mechanisms per se.…”

Section: Discussionmentioning

confidence: 99%

Exploring the chemistry and evolution of the isomerases

Cuesta

Rahman

Thornton

2016

Self Cite

Isomerization reactions are fundamental in biology, and isomers usually differ in their biological role and pharmacological effects. In this study, we have cataloged the isomerization reactions known to occur in biology using a combination of manual and computational approaches. This method provides a robust basis for comparison and clustering of the reactions into classes. Comparing our results with the Enzyme Commission (EC) classification, the standard approach to represent enzyme function on the basis of the overall chemistry of the catalyzed reaction, expands our understanding of the biochemistry of isomerization. The grouping of reactions involving stereoisomerism is straightforward with two distinct types (racemases/epimerases and cis-trans isomerases), but reactions entailing structural isomerism are diverse and challenging to classify using a hierarchical approach. This study provides an overview of which isomerases occur in nature, how we should describe and classify them, and their diversity.T he 3D structure and function of biomolecules are intimately linked. One of the most outstanding attributes of enzymes is their ability to recognize similar molecules, such as isomers, selectively. For example, glutamate racemase catalyzes the interconversion between the isomers L-glutamate and D-glutamate, with the first being one of the 20 amino acids used to build proteins, whereas the second is an essential component of bacterial cell walls (1). Isomers of the same drug are often distinguished; for example, the tragic story of thalidomide unveiled how subtle changes in the spatial arrangement of atoms can have drastic consequences in their biological effect (2).The isomerases, which catalyze these interconversions, are involved in the central metabolism of most living organisms and have important applications in organic synthesis, biotechnology, and drug discovery (3-5). In comparison to other classes, isomerases are a small class involving unimolecular reactions, which are easy to analyze manually. The study of the biological mechanisms of isomerases provided fundamental insights into the electrostatic principles of enzyme catalysis (6) and helped to reveal the connection between host-parasite interactions and cancer (7). The challenges of automatically detecting stereoisomerization in reactions also make their chemistry technically interesting (8-11).A standard description of the biological function of genes and proteins is essential to interpret and report the outcome of sequencing initiatives. Scientists have traditionally developed elaborate classification systems to group functions in a hierarchical manner. Among the existing approaches, enzyme function is probably the best described at the molecular level, due to the longstanding effort of a team of enzymologists from the Enzyme Commission (EC) of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) to classify enzyme function systematically. The EC classification is the most widely used system and uses...

“…Propensity of a particular residue is defined to be the ratio of its frequency among all template residues for the protein structures in the experiment to its frequency within all residues for the protein structures (82). To get better insight, we used log propensity, which is defined to be the logarithm of the calculated ratio.…”

Section: Methodsmentioning

confidence: 99%

Prediction and experimental validation of enzyme substrate specificity in protein structures

Amin

Erdin

Ward³

et al. 2013

Structural Genomics aims to elucidate protein structures to identify their functions. Unfortunately, the variation of just a few residues can be enough to alter activity or binding specificity and limit the functional resolution of annotations based on sequence and structure; in enzymes, substrates are especially difficult to predict. Here, large-scale controls and direct experiments show that the local similarity of five or six residues selected because they are evolutionarily important and on the protein surface can suffice to identify an enzyme activity and substrate. A motif of five residues predicted that a previously uncharacterized Silicibacter sp. protein was a carboxylesterase for short fatty acyl chains, similar to hormone-sensitive-lipase-like proteins that share less than 20% sequence identity. Assays and directed mutations confirmed this activity and showed that the motif was essential for catalysis and substrate specificity. We conclude that evolutionary and structural information may be combined on a Structural Genomics scale to create motifs of mixed catalytic and noncatalytic residues that identify enzyme activity and substrate specificity. A s the list of known genes grows exponentially, the elucidation of their function remains a major bottleneck and lags far behind the production of sequences (1-5). The best approach remains to search computationally for functionally characterized sequence homologs, ideally with greater than 50% sequence identity (6). Binding specificity, however, is sensitive to subtle amino acid differences, and the transfer of substrate between related enzymes is prone to errors when sequence identity is below 65-80% (7-9). These thresholds vary from case to case: Some orthologs will maintain identical functions down to 25% sequence identify (9), whereas paralogs can take on highly diverse activities (10). Other difficulties that plague annotation transfer between homologs are that individual small molecules may each bind to multiple and distinct molecular pockets (11), that different residues can support similar chemistries (12), and that activity can vary even when catalytic residues are conserved (13-18). To raise annotation accuracy, Structural Genomics (19) made structural information widely available and spurred the development of annotation methods dependent on local chemical and physical environments (20), sequence and structural comparisons (21), or 3D templates (22). In the case of the latter, these methods search between proteins for local structural similarities over a few signature residues that represent the telltale parts of a functional site, so-called "3D templates" (3,14,18,(22)(23)(24). The residue composition of 3D templates is critical, however, and derived from experiments (25) or from analyses of functional sites and determinants (14,15,26). The sensitivity and specificity of template-based annotations still needs to be established experimentally (27, 28), but retrospective controls suggest they often predict enzyme catalytic activity (14,16,17,29,30...