Figure 1.
GUS catalyzes the hydrolysis of a glucuronic acid-containing glycosaminoglycan to form two products, glucuronic acid and an amino sugar (acetylglucosamine in this reaction). pNP-GLU is used as the substrate instead of a glycosaminoglycan because para-nitrophenolate absorbance allows for spectroscopic monitoring of activity in experimental studies. Experimental activity measurements for GUS variants are used for verifying correlations between activity and IE.
Figure 2.
Comparison between ground state, hypothetical TS, and TSA for pNP-GLU and pNP-GAL.
Differences between pNP-GLU (A) and pNP-GAL (B) include reversal of the stereospecificity of the C4 carbon and replacement of a carboxylic acid (pNP-GLU) at the C5 carbon with an alcohol (pNP-GAL). The previously-suggested [52], [53] TSA for pNP-GLU, 1,5-glucarolactone (D), resembles the proposed TS (C) in terms of charge distribution and stereospecificity of the carbohydrate. In contrast to the proposed TS structure, the TSA lacks the para-nitrophenyl (pNP) moiety and a hydrogen atom from the C1 carbon. In addition, the TSA (D) differs from pNP-GLU (A) by assuming a more flattened sugar ring geometry (see Figure S1 for dihedral angles) and partial positive charge at the anomeric carbon. The TSA for pNP-GAL, 1,5-galactonolactone (E), is similar to 1,5-glucaronolactone (D). The differences between 1,5-galactonolactone and 1,5-glucaronolactone are identical to the differences between pNP-GAL and pNP-GLU.
Figure 3.
Active site geometry and restrained catalytic contacts.
The active site is depicted in a ball-and-stick representation (C = black, O = red, N = blue, H = white). The nonbonded interactions seen reflect the distances restrained (as listed in Table 1). Key catalytic residues are labeled by their one-letter amino acid abbreviation followed by their position number. para-nitrophenyl- β, D-glucuronide (pNP-GLU) is labeled by the abbreviation “PNP” (see Figure 1). Atoms involved in restraints are labeled, along with interatomic distances.
Table 1.
NOE restraints applied during CHARMM energy minimization.
Figure 4.
Proposed catalytic reaction mechanism of GUS from in vacuo QM calculations (Text S1).
In the first step, substrate binds to the active site of GUS. Next, the lone pair on the glycosidic bond attacks the proton of E413 (A). This forms a hypothetical TS (B) with the glycosidic bond partially broken. The glycosidic bond is fully cleaved, releasing para-nitrophenolate and forming a carbocation intermediate (C). The electrons on the anionic E504 then attack the anomeric carbon, resulting in a hypothetical TS (D) where the carbocation and E504 are electrostatically attractive. A covalent intermediate (E) is formed between the carbohydrate moiety of pNP-GLU and E504. Presumably, in the next step, the basic E413 attacks a proton of a water molecule. The resulting hydroxide anion attacks the anomeric carbon to yield the product of the reaction. The two catalytic residues are regenerated for further turnover.
Figure 5.
Ground state computational IES for pNP-GLU versus the natural logarithm of experimental KM.
IEs were calculated using IPRO, and experimental data was obtained from literature [48], [49], [57]. Each numbered label corresponds to a single variant enzyme with multiple amino acid substitutions from wild-type (WT). Calculated IEs at the ground state are consistent with the observed changes in KM for GUS mutants (R2 = 0.960). Figure S2 shows the distribution of the trajectory-best IEs whose average forms each data point.
Figure 6.
Qualitative GUS free energy diagram based upon in vacuo QM calculations.
The free energy of each intermediate within the dashed box is based on its potential energy, as calculated using QM. Intermediates found using QM and proposed TSs are also labeled according to Figure 4 (italicized, above curve). The energy barrier between states C and D is nearly barrier-less. The free energy values along the remainder of the curve are purely hypothetical. Each intermediate is labeled according to the convention used in Equation 3. Based on the known and hypothesized free energies, the reaction of the Michaelis complex to form the first intermediate (k2, as written in Equation 3) is rate-limiting. Thus, the proposed TS for the entire reaction (E·TS) and its corresponding energy barrier (ΔG‡) are labeled.
Figure 7.
Computationally-determined IETSA for pNP-GLU versus experimental ln(kcat/KM).
Data was collected as described in Figure 5. Enzyme variants with higher catalytic efficiency (kcat/KM) have a stronger affinity for 1,5-glucarolactone (R2 = 0.864). See also Figure S3.
Figure 8.
Scaled difference between IETSA and IES for pNP-GLU versus the natural logarithm of kcat.
Data was obtained as detailed in the caption of Figure 5. Scaling is required because of the non-quantitative nature of the energy calculations. With scaling, it is apparent that the turnover number increases as the difference becomes more negative. These results suggest that as the enzyme interacts with the TS more strongly, the turnover number increases (R2 = 0.854).
Figure 9.
pNP-GLU IES correlation with catalytic efficiency.
Data was obtained as described for Figure 5. No significant correlation is observed (R2 = 0.545) between IE with pNP-GLU and ln(kcat/KM). If GUS catalysis was primarily achieved through reactant destabilization, a positive slope would have been expected.
Figure 10.
Correlation between pNP-GAL IETSA and ln(kcat/KM).
The correlation found here is significantly lower than the one found for pNP-GLU (see Figure 7) primarily due to mutant T509S. See also Figure S4.
Figure 11.
Distribution of amino acids in a sequence alignment for all β-glucuronidases.
The sequence alignment was performed over all β-glucuronidases (as identified using BRENDA) using the Clustal-Omega algorithm. 181 unique sequences were used during the alignment. Design position numbers indicate the position within GUS, and the one-letter abbreviation for WT E. coli β-glucuronidase is provided at each position. Only amino acids observed >1% of the time at a given position are shown since smaller bars were difficult to decipher. With the exception of H162, the E. coli WT residue is the amino acid most frequently observed in the alignment.
Table 2.
Permitted amino acids at each design position.
Figure 12.
Distribution of amino acids for top 50 GUS mutants enhancing enzyme catalytic parameters of pNP-GLU.
The libraries were designed to optimize (A) KM, (B), kcat/KM, and (C) kcat. Design position numbers indicate the position within GUS, and the one-letter abbreviation for WT GUS is provided.
Table 3.
Top 10 mutants identified using OptZyme for optimizing KM, kcat/KM, and kcat for pNP-GLU.
Figure 13.
Distribution of amino acids for top 50 GUS mutants enhancing enzyme catalytic parameters of pNP-GAL.
The libraries were designed to optimize (A) KM, (B), kcat/KM, and (C) kcat. Design position numbers indicate the position within GUS, and the one-letter abbreviation for WT GUS is provided.
Table 4.
Top 10 mutants identified using OptZyme for optimizing KM, kcat/KM, and kcat for pNP-GAL.