Methods for assaying homocysteine

A method for assaying homocysteine (Hcy), S-adenosylhomocysteine (SAH) or adenosine in a sample, which method comprises:

• a) contacting a sample containing or suspected of containing Hcy, SAH or adenosine with a mutant SAH hydrolase derived from a SAH hydrolase, wherein said SAH hydrolase is encoded by a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, and SEQ ID NO:188;
• wherein said mutant SAH hydrolase has binding affinity for Hcy, SAH or adenosine but has attenuated catalytic activity, and wherein said binding affinity and/or said attenuated catalytic activity of said mutant SAH hydrolase is caused by a mutation in said mutant SAH hydrolase's catalytic site, its binding site for NAD+, NADH, Hcy, SAH, adenosine, or a combination thereof;
• and wherein the mutant SAH hydrolase:
• i) has a mutation in an amino acid residue that participates in catalysis or that is directly interacting with NAD+, NADH, Hcy, SAH or adenosine, or ii) has a mutation in an amino acid residue that is adjacent to an amino acid residue that participates in catalysis or that is directly interacting with NAD+, NADH, Hcy, SAH or adenosine, wherein the mutation in said mutant SAH hydrolase corresponding one or more amino acid positions selected from the group consisting of 53, 54, 57, 59, 80, 83, 121, 131, 134, 155, 157, 158, 159, 181, 190, 191, 214, 221, 226, 235, 240, 248, 263, 269, 285, 292, 301, 309, 322, 347, 351, 353, 361, 362, 379, 386, 388, 398, 401, 407, 409, 424, 425, 426, 427, 428, 429, 430, 431, and 432 SEQ ID NO:1 in said and
• b) detecting binding between Hcy, SAH or adenosine with said mutant SAH hydrolase, whereby the presence or amount of Hcy, SAH or adenosine in said sample is assessed.