The characteristic feature profiles of catalytic residues

3.2.1 Sequence conservation profile

Catalytic sites are clearly more conserved than other residues; it can be shown in Figure 2(A). The average conservation score of catalytic sites is 0.09. Since the catalytic sites in proteins are all have important function, it would not be easily substituted by other amino acids. Figure 3 shows the conservation score trend of 11 amino acids respectively. The trends of the performance of these amino acids appear to be similar.

3.2.2 Secondary structure profile

Figure 2(B) and Figure 2(C) show the secondary structure distribution of catalytic sites compared with all residues in the dataset. Catalytic sites prefer to locate on the coil regions (about 50%) than other types. On the contrary, catalytic sites not favor to occur in helix regions (only 23%). This is different from the distribution of all residues. When we use the eight states structural categories followed by DSSP, we can found out that catalytic sites are especially prefer to occur in  ladder (E) and undefined (U) regions (Figure 2(C)).

Make it more clearly, in Figure 4 and Figure 5; we analyze the 11 amino acid individually. The catalytic sites of threonine and tyrosine have different distribution with

11

others; they do not often locate on  ladder. It may because both of them have the hydroxyl group on the side-chain. Furthermore, cysteine, whose side-chain has thiol group, also not prefers to occur in  ladder region. Oppositely, aspartate and glutatmate, who have negatively carboxyl group on the side-chain, are more prefer to locate on  ladder regions.

The remaining six amino acids have similar distribution of secondary structure.

3.2.3 Relative Solvent Accessibility (RSA) profile

Figure 2(D) and Figure 2(E) show the relative solvent accessibilities profiles of catalytic sites compared with all residues in the dataset. As we have mentioned in our method, we use binary and ternary model to analyze the distribution of catalytic sites. The 67% of catalytic sites are more buried residues in protein structures (Figure 2(D)). While in ternary model of relative solvent accessibility, only 11% of all catalytic sites are fully exposed. This result consistent with other study which shows that the catalytic site is often occurred in a large and deep cleft or cavity ²³.

We analyzed 11 amino acids respectively, as shown in Figure 6 and Figure 7. The side chain of cysteine is thiol group, surprisingly; its distribution between catalytic sites and all residues is quite different with other amino acids. The RSA trend of catalytic sites or all residues of cysteine are exceptionally similar, both of them tend to have more buried to solvent. The reason for that might because the thiol group of cysteine is the most reactive side chain found amongst the 20 naturally amino acid residues. However, the exposed frequency of catalytic sites of cysteine is unusual higher than all residues. It may due to that the side chain of cysteine is prefer to form disulfide bonds, which is a strong covalent bond and adopted in solution.

12

3.2.4 Rigidity profile

In this study we use three kind features (i.e., zB-factor, CM, WCN) to represent a protein structural rigidity. The B-factor is often used to measure residue flexibility, the smaller value is, and the less flexibility is. The smaller WCN value a residue is means that it locates on more crowded environment. The CM value represents whether a residue is close to its structural center or not.

Figure 2(F) compares the zB-factor of the catalytic sites with that of all residues. There are around 81% of catalytic sites with zB-factor ≦ 0, compared with 54% of all residues.

Figure 2(G) shows that the WCN of catalytic sites compared with all residues. There are around 90% of catalytic sites with WCN ≦ 0, compared with 14% of all residues. Moreover, the CM of catalytic sites compared with all residues is shown in Figure 2(H). It should be noted that there are about 94% of catalytic sites with CM ≦ 0, compared with 13% of all residues. No matter which feature of these three can suggest that catalytic residues tend to be more rigidity, it means catalytic sites often held in fixed place in enzyme than all residues.

However, we further shows that WCN and CM can perform a much better result than zB-factor. If we use a cutoff value ≦ 0, the WCN and CM will contain about more than 90%

of the catalytic residues. CM shows that the catalytic sites are usually closet to the protein centroid. The lower WCN means the catalytic sites tend to lie in the more packed regions than other residues do. According to our results, CM and WCN can play an expressive role in determine the catalytic sites, since the refined B-factor easily affecting by factors like temperature, crystallization conditions or structural refinement.

zB-factor, CM, WCN plots for individual amino acid types can be seen in Figure 8,9,10, respectively. The charged side-chain catalytic residues are easier to be differentiated with all residues. Nevertheless, figure 9(F)(G) and figure10 (F)(G) show that the catalytic sites of

13

serine and threonine, which has polar side chain, especially more prefer the environment which is more crowed and more center than tyrosine. It is reasonable that tyrosi²⁴ne needs more space for its larger side chain.

It is worth noting that, our results are consistent with previous study²⁴. Since 1894, Emil Fisher proposed that the catalytic site has a specific geometric shape that is features to plot 2D-profiles (Figure 11).

Figure 11(A) shows the relationship between conservation score and relative solvent accessibility. Figure 11(B) shows the relationship between conservation score and normalized B-factor. Figure 11(C) shows the relationship between conservation score and centriod model and figure 11 (D) shows the relationship between conservation score and weighted contact number. The conservation score and RSA in catalytic sites are usually low, but conservation score has no readily observable connection with RSA (Figure 11 (A)).

However, using the zB-factor, CM, or WCN may help the conservation score to have a better discrimination between catalytic sites and all residues as shown in Figure 11 (B) (C) (D).

Next, we discuss about the relative solvent accessibility combined with other features, which are zB-factor, CM, and WCN, the values of all features tend to be low as shown in figure 11 (E)(F)(G). In this case, we might say that if a residue’s RSA value lower than 0.36 (means intermediate or buried) ²⁰ and CM/WCN value lower than 0, it usually located on the catalytic sites.