Origin of the Different pH Activity Profile in Two Homologous Ketosteroid Isomerases

Abstract

Two homologous ⌬5 -3-ketosteroid isomerases from Comamonas testosteroni (TI-WT) and Pseudomonas putida biotype B (PI-WT) exhibit different pH activity profiles. TI-WT loses activity below pH 5.0 due to the protonation of the conserved catalytic base, Asp-38, while PI-WT does not. Based on the structural analysis of PI-WT, the critical catalytic base, Asp-38, was found to form a hydrogen bond with the indole ring NH of Trp-116, which is homologously replaced with Phe-116 in TI-WT. To investigate the role of Trp-116, we prepared the F116W mutant of TI-WT (TI-F116W) and the W116F mutant of PI-WT (PI-W116F) and compared kinetic parameters of those mutants at different pH levels. PI-W116F exhibited significantly decreased catalytic activity at acidic pH like TI-WT, whereas TI-F116W maintained catalytic activity at acidic pH like PI-WT and increased the k cat /K m value by 2.5-to 4.7-fold compared with TI-WT at pH 3.8. The crystal structure of TI-F116W clearly showed that the indole ring NH of Trp-116 could form a hydrogen bond with the carboxyl oxygen of Asp-38 like that of PI-WT. The present results demonstrate that the activities of both PI-WT and TI-F116W at low pH were maintained by a tryptophan, which was able not only to lower the pK a value of the catalytic base but also to increase the substrate affinity. This is one example of the strategy nature can adopt to evolve the diversity of the catalytic function in the enzymes. Our results provide insight into deciphering the molecular evolution of the enzyme and creating novel enzymes by protein engineering.Isozymes carry out the same enzymatic reaction yet exhibit different properties such as substrate affinity, preference of different coenzyme, stability, optimal pH, etc. Even if the sequence differences of those isozymes are evident, the origin of different properties is mostly not clear at the molecular level. Identification of functional divergence on the mechanistic basis is important to understand how the enzyme as a molecular machine can be evolved in the biological system and to rationally modify the enzyme function with desired physical and catalytic properties (1). In the post-genomic era, protein sequences and high resolution protein structures are being accumulated rapidly, but the remarkable catalytic mechanism of enzymes as well as the catalytic strategies utilized by nature to evolve new catalytic functions remain to be understood at the molecular level (2). The understanding of the structural basis for different properties of isozymes and their catalytic diversity could provide the insights into not only discovering the properties of the enzymes from their protein sequences or structural data but also creating novel enzymes by protein engineering.⌬ 5 -3-Ketosteroid isomerase (KSI, 1 EC 5.3.3.1) is one of the most proficient enzymes catalyzing an allylic isomerization reaction at a rate comparable to the diffusion-controlled limit by an intramolecular transfer of a proton (Scheme 1) (3-8). Although KSIs from two different bacterial sources, Comamon...

Evolutionary specialization of a tryptophan indole group for transition-state stabilization by eukaryotic transglutaminases

“…By analogy with papain and other enzymes (e.g., tyrosyl-tRNA synthetase; ref. 26), a ⌬⌬G T ‡ of Ϸ4 kcal͞mol might be attributed to loss of a hydrogenbonding interaction (27,28) between the Trp-241 side chain and the substrate oxyanion. Trp-241 might similarly stabilize oxyanion intermediate 2 in the deacylation step (Fig.…”

Evaluation of the Energetics of the Concerted Acid–Base Mechanism in Enzymatic Catalysis: The Case of Ketosteroid Isomerase

“…18,19 The question is whether the OAH’s function is to serve as a general acid (donating a proton to the intermediate) or as an electrophile (donating hydrogen bonds and stabilizing a charged intermediate). KSI’s capacity to abstract a weakly-acidic α-proton from its steroid substrate (p K a = 12.7 20 ) with its very weak general base (p K a = 3.75 21 ) has previously been attributed to concerted proton transfer, path (ii) in Figure 1B. 18,22–25 …”

Quantitative, directional measurement of electric field heterogeneity in the active site of ketosteroid isomerase

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