Human aorta lysyl oxidase

Lysyl oxidase (LOX, EC 1.4.3.13) is a copper-dependent amine oxidase expressed and secreted by fibrogenic cells. It catalyzes a critical step in the crosslinking of collagen and elastin by oxidative deamination of the e-amino group of lysine and hydroxylysine to peptidyl a-aminoadipic-d-semialdehyde. The general equation for the lysyl oxidase catalyzed reaction is:

RCH₂NH₂ + O₂ + H₂O ® RCHO + NH₃ +H₂O₂

The resulting aldehyde molecules spontaneously condense with neighboring amino groups or other peptidyl aldehydes to form covalent crosslinks in fibrillar collagens and elastin. These lysine-derived crosslinkages serve to insolubilize and stabilize collagen molecule. For this reason, LOX plays a key role in the morphogenesis and repair of connective tissues of the cardiovascular, respiratory, skeletal, and other systems of the body. More recently, several other functions have also been attributed to LOX including roles in tumor suppression, cellular senescence, developmental control, and chemotaxis.

The following are active areas of research for lysyl oxidase:

Elucidation of histidine residues involved in copper-binding

The incorporation of Cu(II) into LOX is of paramount importance. It has been postulated that there is a peptide region in LOX that contains histidines that bind copper and this region is denoted as a so-called copper-binding talon. Based on published data, the histidines most likely to be involved are histidine 289, histidine 292, histidine 294, histidine 296, and histidine 303.

Our laboratory is currently pursuing three of these histidines as potential copper ligands.

Role of lysine in the LTQ crosslink

One of the features that set the LTQ cofactor (Figure 1A) apart from the cofactor of other amine oxidases (Figure 1B) is the self-processed crosslink of lysine 320 with the oxidized tyrosine 355.

Figure 1 Lysyl tyrosyl quinone (LTQ) of lysyl oxidase (A) and 2,4,5-trihydroxyphenylalanine quinone (TPQ) of other amine oxidase enzymes (B).

The biogenesis pathway for the formation of TPQ has been extensively studied and a plausible mechanism for the formation of TPQ has been elucidated. In contrast, very little is known about the biogenesis of LTQ, although it has been postulated that the mechanisms are similar through the formation of the intermediate dopaquinone (DPQ) (Scheme 1). At this stage, lysine is believed to crosslink with DPQ thus preventing the free rotation needed to proceed to TPQ and directing the biogenesis to LTQ through pathway A in Scheme 1. However, since this pathway has not been unambiguously elucidated, it is plausible that LTQ formation proceeds through pathway B, leading to TPQ and then, through a series of yet to be determined steps, LTQ is formed from a TPQ intermediate. We are attempting to elucidate the pathway to LTQ using a combination of kinetic and spectrophotometric (UV-Vis) experiments, as well as stopped-flow experiments.

Scheme 1 Possible pathways leading to the formation of LTQ

Determination of the catalytic base of lysyl oxidase

Lysyl oxidase catalyzes the oxidation of primary amines via a ping-pong mechanism (Scheme 2). Following the initial Schiff base formation the bound substrate undergoes a rate-limiting general base proton abstraction. This general base has been postulated to be a histidine residue in the vicinity of the active site; however, the identity of this histidine residue has yet to be determined. Our focus is to determine if a histidine is the catalytic base and, if so, which one.

Scheme 2 Catalytic cycle of lysyl oxidase