Structure and catalysis of acylaminoacyl peptidase: closed and open subunits of a dimer oligopeptidase

Abstract

Acylaminoacyl peptidase from Aeropyrum pernix is a homodimer that belongs to the prolyl oligopeptidase family. The monomer subunit is composed of one hydrolase and one propeller domain. Previous crystal structure determinations revealed that the propeller domain obstructed the access of substrate to the active site of both subunits. Here we investigated the structure and the kinetics of two mutant enzymes in which the aspartic acid of the catalytic triad was changed to alanine or asparagine. Using different substrates, we have determined the pH dependence of specificity rate constants, the rate-limiting step of catalysis, and the binding of substrates and inhibitors. The catalysis considerably depended both on the kind of mutation and on the nature of the substrate. The results were interpreted in terms of alterations in the position of the catalytic histidine side chain as demonstrated with crystal structure determination of the native and two mutant structures (D524N and D524A). Unexpectedly, in the homodimeric structures, only one subunit displayed the closed form of the enzyme. The other subunit exhibited an open gate to the catalytic site, thus revealing the structural basis that controls the oligopeptidase activity. The open form of the native enzyme displayed the catalytic triad in a distorted, inactive state. The mutations affected the closed, active form of the enzyme, disrupting its catalytic triad. We concluded that the two forms are at equilibrium and the substrates bind by the conformational selection mechanism.

SCHEME 1

FIGURE 1.

pH dependence in the reactions of Ac-Phe-Nap with ApAAP and its variants, D524A and D524N. A, the substrate is Ac-Phe-Nap. Circles, full circles, and crosses, refer to ApAAP, the D524A variant, and the D524N variant, respectively. The lines for the wild-type and the D524A variant represent bell-shaped and doubly sigmoid curves, respectively. The parameters for the curves are shown in Table 1. B, the substrate is Abz-Ala-Leu-Phe-Gln-Gly-Pro-Phe(NO₂)-Ala. The signs for the enzymes are identical with those shown in A.

FIGURE 2.

pH dependence of intrinsic fluorescence of ApAAP. Fluorescence intensity at 0.372 μm enzyme was measured at 70 °C and shown in arbitrary units. The excitation and emission wavelengths are 280 and 335 nm. A bell-shaped curve was fit to the points. The insert shows the fit of a sigmoid curve with a pK_a of 9.96 ± 0.05 at the alkaline side.

FIGURE 3.

Determination of K_s for the Abz-Ala-Leu-Phe-Gln-Gly-Pro-Phe(NO₂)-Ala-APAAP(S445A/D524A) complex. 0.2 μm substrate was titrated with increasing amounts of enzyme.

FIGURE 4.

pH dependence of 1/K_i for the complex of Ac-Phe-OH formed with ApAAP and its variants. The circle and the cross represent the wild-type and the D524N variant, respectively. The full circle refers to the D524A variant. The parameters for the curves are shown in Table 3. The first-order rate constants were measured with Ac-Phe-Nap.

FIGURE 5.

Overall conformation of the ApAAP structures. A, molecular surface representation of the known ApAAP dimer structures (monomers are shown in blue and green; hydrolase domains are in darker colors). Previously published structures (PDB ID codes 1VE6, 1VE7, 2HU5, 2HU7, and 2HU8) show the closed/closed conformation. Three of the present structures are in the open/closed conformation (the interdomain opening is marked with a red arrow), and one is in the open/open conformation. B, Cα-Cα distances of residues involved in interdomain hydrogen bonds are marked with orange lines, whereas those in the hinge region are represented with red lines. Most of the hydrogen bonds of the closed structure (left) are broken in the open structure (right); in contrast, the hydrogen bonds of Asp³⁷⁶ (hinge region, red lines) are preserved. C, the superposition of the open structures reveals their similarity (all eight of the open monomers of the four crystal structures are shown). The schematic representations of the open structures are color-ramped blue to red from low to high residual B-factors. Left, loop-(83–88) and loop-(551–560) (containing the catalytic His⁵⁵⁶) show high mobility in all of the open structures. Right, the conformation of the loop containing His⁵⁵⁶ (Cα atom shown with a sphere) is also changed significantly. The closed structure is shown in gray as reference with the catalytic triad in stick representation.

FIGURE 6.

Rearrangement of the active site caused by structure opening or the D524A and D534N mutations. Conformations and hydrogen bond networks of the loops holding the active site residues in the closed and open structures are shown in A and B, respectively. Loop-(551–560) holding His⁵⁵⁶ and loop-(521–529) holding Ala⁵²⁴ are shown in magenta and cyan, respectively. Ser⁴⁴⁵ is shown with spheres in the background. The propeller domain is shown in lighter gray with loops 43–46 and 83–88 in yellow. Note that the conformation and interactions of loop-(521–529) are similar in the mutant and native forms, whereas those of loop-(551–561) vary. C and D show the effect of mutation on the active site of the closed, intact structures. C, superposition of the D524A mutant and wild-type structures. D, superposition of the D524N mutant and wild-type structures. The catalytic histidine is in dual conformation. The loops of the mutant forms are colored as on A and B. Ser⁴⁴⁵, green with orange hydrogen bonds. The native structure is transparent gray with light green hydrogen bonds. Although the backbone conformation is unchanged in the mutant forms, the His⁵⁵⁶ side chain is rotated in a catalytically less favorable position, and the Ser-His hydrogen bond is lost. The distortion effect is more enhanced in the D524N form.

SCHEME 2