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A lyase is an enzyme classified by cleavage through means other than hydrolysis or oxidation. Such reactions commonly eliminate a group to create a double bond or a ring; the reverse direction adds a group across the corresponding structural feature. Lyases form EC class 4, with subclasses organized partly by the bonds affected.^[ref]

The involvement of water alone does not make a reaction hydrolysis. Fumarate hydratase, also called fumarase, interconverts fumarate plus water with the (S) form of malate. In the water-removing direction, it generates the double bond of fumarate. In the reverse direction, water adds across that bond.^[ref] The classification concerns the transformation, not merely whether water appears somewhere in its equation.

An invented bookkeeping example begins with $5\mu \mathrm{mol}$ of (S)-malate. If $3\mu \mathrm{mol}$ undergoes the water-eliminating conversion, $2\mu \mathrm{mol}$ malate remains and $3\mu \mathrm{mol}$ fumarate is formed. No carbon atom is removed from the four-carbon skeleton in this reaction. This is a stipulated extent, not a prediction of the position of chemical equilibrium or the reaction speed.

The distinction can also arise in a multistep reaction. Ribonuclease T1 first produces a cyclic phosphate end by a phosphorus–oxygen lyase step and subsequently hydrolyses that cyclic product. Its classification emphasizes the primary cleavage reaction, even though the overall process also includes hydrolysis.^[ref]

Thus a lyase classification gives useful chemical information but does not uniquely specify a protein's structure, substrate range or physiological role. The actual reaction and its experimentally observed direction remain essential to interpreting the enzyme's name.

Ribonuclease T1 is a guanosine-selective ribonuclease: it cleaves susceptible phosphodiester bonds on the 3′ side of guanosine residues in single-stranded RNA. Its two-stage chemistry first creates an RNA cyclic phosphate end and a 5′-hydroxyl end, then converts the cyclic end to a 3′-phosphate end by hydrolysis.^[ref]

The current classification is EC 4.6.1.24, a lyase entry. The former EC 3.1.27.3 entry was transferred in 2020 because the primary cleavage step is a phosphorus–oxygen lyase reaction. Merely describing the final overall reaction as hydrolysis hides that first step.^[ref]

An invented sequence example makes its selectivity concrete. Read ACGUAAGCU from 5′ to 3′ and suppose all eligible internal bonds are accessible and completely cleaved. Cutting after each G gives ACG|UAAG|CU: fragments of three, four and two nucleotides, respectively. The nine nucleotides are redistributed among three fragments rather than disappearing. For a linear chain, $k$ distinct internal cuts produce $k+1$ fragments.

The letters alone do not guarantee that a folded RNA exposes every candidate bond. Sequence preference and physical accessibility jointly affect a cleavage pattern; the toy calculation explicitly assumes complete accessibility.

This selectivity also made ribonuclease T1 useful in historical sequence comparisons. A 1977 study characterized 16S ribosomal RNA from ten methane-producing microbial species through the fragments produced by T1 digestion, then compared the resulting catalogs.^[ref] Selective cleavage converted a large molecule into a collection of experimentally comparable sequence pieces.