Catalytic Mechanism of Collagen Hydrolysis by Zinc(II)-Dependent Matrix Metalloproteinase-1

Abstract

Human matrix metalloproteinase-1 (MMP-1) is a zinc(II)-dependent enzyme that catalyzes collagenolysis. Despite the availability of extensive experimental data, the mechanism of MMP-1-catalyzed collagenolysis remains poorly understood due to the lack of experimental structure of a catalytically productive enzyme-substrate complex of MMP-1. In this study, we apply molecular dynamics and combined quantum mechanics/molecular mechanics to reveal the reaction mechanism of MMP-1 based on a computationally modeled structure of the catalytically competent complex of MMP-1 that contains a large triple-helical peptide substrate. Our proposed mechanism involves the participation of an auxiliary (second) water molecule (wat₂) in addition to the zinc(II)-coordinated water (wat₁). The reaction initiates through a proton transfer to Glu219, followed by a nucleophilic attack by a zinc(II)-coordinated hydroxide anion nucleophile at the carbonyl carbon of the scissile bond, leading to the formation of a tetrahedral intermediate (IM2). The process continues with a hydrogen-bond rearrangement to facilitate proton transfer from wat₂ to the amide nitrogen of the scissile bond and, finally, C-N bond cleavage. The calculations indicate that the rate-determining step is the water-mediated nucleophilic attack with an activation energy barrier of 22.3 kcal/mol. Furthermore, the calculations show that the hydrogen-bond rearrangement/proton-transfer step can proceed in a consecutive or concerted manner, depending on the conformation of the tetrahedral intermediate, with the consecutive mechanism being energetically preferable. Overall, the study reveals the crucial role of a second water molecule and the dynamics for effective MMP-1-catalyzed collagenolysis.

Figure 1

Structure of the MMP-1·THP complex. The zoomed-in panel on the left shows the catalytic zinc(II) coordinates to the scissile bond carbonyl Gly775, His218, His222, and His228, and a water molecule (wat₁) polarized by the base Glu219.

Figure 2

QM region used in the QM/MM calculations.

Figure 3

(A) Residues involved in hydrogen bonding between THP and the CAT and HPX domains in the RC. (B) Zoomed-in view of the hydrogen bonding interactions between the L-chain residues Gln774, Ala777, and Gly778 (orange) and the CAT domain residues (tan). The hydrogen atoms not involved in interactions are hidden for clarity.

Figure 4

Stationary point geometries for proton abstraction by Glu219. The corresponding distances (Å) are given in the blue boxes next to each structure. Hydrogen atoms of the substrate other than the scissile bond residues are hidden for clarity.

Scheme 1. Proposed Reaction Mechanism of MMP-1 with the Participation of Two Water Molecules (wat₁ and wat₂)

Figure 5

Potential energy profile of the MMP-1 reaction mechanism. The relative B3 (B2 + ZPE) energies are shown in red. The yellow oval-shaped denotation indicates that MD simulations were run on both RC and IM2. Energy values are in kcal/mol.

Figure 6

Stationary point geometries for the nucleophilic attack. The corresponding distances (Å) are given in the blue box next to each structure. Hydrogen atoms other than the scissile bond ones are hidden for clarity.

Figure 7

TS stabilizing SCS residues (yellow) of the rate-determining nucleophilic attack. The hydrogen atoms not involved in the interactions are hidden for clarity.

Figure 8

Potential energy profile (kcal/mol) for hydrogen-bond rearrangement and proton transfer. (A) Snapshots 5818 (black), 6594 (pink), and 4642 (blue) show consecutive hydrogen-bond rearrangement and proton transfer. (B) Snapshot 8234 (green) shows a concerted mechanism. The relative B3 energies are shown for the four snapshots.

Figure 9

Stationary point geometries of the hydrogen-bond rearrangement of the lowest barrier snapshot (5818). The corresponding distances (Å) are given in the blue box next to each structure. Hydrogen atoms of the substrate other than the scissile bond residues are hidden for clarity.

Figure 10

Stationary point geometries of proton transfer (snapshot 5818). The corresponding distances (Å) are given in the blue boxes next to each structure. Hydrogen atoms of the substrate other than the scissile bond residues are hidden for clarity.

Figure 11

Tetrahedral intermediates (IM2′) from (A) snapshot 5818 (representing snapshots 5818, 6594, and 4642) and (B) snapshot 8234, demonstrating the hydrogen bonding interactions in the catalytic site leading to the consecutive and concerted hydrogen-bond rearrangement/proton-transfer reaction step. The hydrogens of the carbon atoms are hidden for clarity.

Figure 12

TS stabilizing residues for the (A) consecutive hydrogen-bond rearrangement and proton transfer in snapshots 5818, 6594, and 4642 and (B) concerted mechanism in snapshot 8234 of the IM2'.

Figure 13

Stationary point geometries of the C–N bond cleavage. The corresponding distances (Å) are given in the blue boxes next to each structure. Hydrogen atoms of the substrate other than the scissile residues are hidden for clarity.