Comparative kinetics of cofactor association and dissociation for the human and trypanosomal S-adenosylhomocysteine hydrolases. 2. The role of helix 18 stability.


Abstract

The S-adenosyl- l-homocysteine (AdoHcy) hydrolases (SAHH) from Homo sapiens (Hs-SAHH) and from the parasite Trypanosoma cruzi (Tc-SAHH) are very similar in structure and catalytic properties but differ in the kinetics and thermodynamics of association and dissociation of the cofactor NAD (+). The binding of NAD (+) and NADH in SAHH appears structurally to be mediated by helix 18, formed by seven residues near the C-terminus of the adjacent subunit. Helix-propensity estimates indicate decreasing stability of helix 18 in the order Hs-SAHH > Tc-SAHH > Ld-SAHH (from Leishmania donovani) > Pf-SAHH (from Plasmodium falciparum), which would be consistent with the previous observations. Here we report the properties of Hs-18Pf-SAHH, the human enzyme with plasmodial helix 18, and Tc-18Hs-SAHH, the trypanosomal enzyme with human helix 18. Hs-18Tc-SAHH, the human enzyme with trypanosomal helix 18, was also prepared but differed insignificantly from Hs-SAHH. Association of NAD (+) with Hs-SAHH, Hs-18Pf-SAHH, Tc-18Hs-SAHH, and Tc-SAHH exhibited biphasic kinetics for all enzymes. A thermal maximum in rate, attributed to the onset of local structural alterations in or near the binding site, occurred at 35, 33, 30, and 15 degrees C, respectively. This order is consistent with some reversible changes within helix 18 but does require influence of other properties of the "host enzyme". Dissociation of NAD (+) from the same series of enzymes also exhibited biphasic kinetics with a transition to faster rates (a larger entropy of activation more than compensates for a larger enthalpy of activation) at temperatures of 41, 38, 36, and 29 degrees C, respectively. This order is also consistent with changes in helix 18 but again requiring influence of other properties of the "host enzyme". Global unfolding of all fully reconstituted holoenzymes occurred around 63 degrees C, confirming that the kinetic transition temperatures did not arise from a major disruption of the protein structure. Study holds ProTherm entries: 23284, 23285, 23286, 23287, 23288, 23289, 23290, 23291, 23292, 23293, 23294, 23295, 23296, 23297, 23298, 23299, 23300, 23301, 23302, 23303 Extra Details: Apo form. ASA mot filled as the structure of 1XBE has not yet been released. S-adenosyl-L-homocysteine hydrolase, Human, unfolding

Submission Details

ID: eBqPi6XB3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:54 p.m.

Version: 1

Publication Details
Li QS;Cai S;Fang J;Borchardt RT;Kuczera K;Middaugh CR;Schowen RL,Biochemistry (2008) Comparative kinetics of cofactor association and dissociation for the human and trypanosomal S-adenosylhomocysteine hydrolases. 2. The role of helix 18 stability. PMID:18393535
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
5AXA 2016-07-27 1.55 Crystal structure of mouse SAHH complexed with adenosine
5AXC 2016-07-27 1.55 Crystal structure of mouse SAHH complexed with 3'-keto aristeromycin
5AXD 2016-07-27 1.6 Crystal structure of mouse SAHH complexed with ribavirin
5AXB 2016-07-27 1.65 Crystal structure of mouse SAHH complexed with noraristeromycin
1LI4 2003-05-20 2.01 Human S-adenosylhomocysteine hydrolase complexed with neplanocin
4PFJ 2014-10-01 2.3 The structure of bi-acetylated SAHH
5W49 2017-06-28 2.4 The crystal structure of human S-adenosylhomocysteine hydrolase (AHCY) bound to oxadiazole inhibitor
3NJ4 2011-05-04 2.5 Fluoro-neplanocin A in Human S-Adenosylhomocysteine Hydrolase
4PGF 2014-10-01 2.59 The structure of mono-acetylated SAHH
5W4B 2017-06-28 2.65 The crystal structure of human S-adenosylhomocysteine hydrolase (AHCY) bound to benzothiazole inhibitor
4YVF 2015-11-25 2.7 Structure of S-adenosyl-L-homocysteine hydrolase
1A7A 1999-04-20 2.8 STRUCTURE OF HUMAN PLACENTAL S-ADENOSYLHOMOCYSTEINE HYDROLASE: DETERMINATION OF A 30 SELENIUM ATOM SUBSTRUCTURE FROM DATA AT A SINGLE WAVELENGTH
1KY5 2002-09-25 2.8 D244E mutant S-Adenosylhomocysteine hydrolase refined with noncrystallographic restraints
1KY4 2002-09-25 2.8 S-Adenosylhomocysteine hydrolase refined with noncrystallographic restraints
1D4F 2001-01-17 2.8 CRYSTAL STRUCTURE OF RECOMBINANT RAT-LIVER D244E MUTANT S-ADENOSYLHOMOCYSTEINE HYDROLASE
1XWF 2005-09-20 2.8 K185N mutated S-adenosylhomocysteine hydrolase
1B3R 1998-12-23 2.8 RAT LIVER S-ADENOSYLHOMOCYSTEIN HYDROLASE
2H5L 2007-04-10 2.8 S-Adenosylhomocysteine hydrolase containing NAD and 3-deaza-D-eritadenine
1K0U 2001-10-17 3.0 Inhibition of S-adenosylhomocysteine Hydrolase by 'acyclic sugar' Adenosine Analogue D-eritadenine

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
96.5 Adenosylhomocysteinase Q3MHL4 SAHH_BOVIN
96.5 Adenosylhomocysteinase P10760 SAHH_RAT
96.5 Adenosylhomocysteinase Q710C4 SAHH_PIG
97.0 Adenosylhomocysteinase P50247 SAHH_MOUSE
98.8 Adenosylhomocysteinase Q4R596 SAHH_MACFA
100.0 Adenosylhomocysteinase P23526 SAHH_HUMAN