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Eukaryotic Cell, December 2006, p. 2014-2023, Vol. 5, No. 12
1535-9778/06/$08.00+0     doi:10.1128/EC.00146-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Activity of a Second Trypanosoma brucei Hexokinase Is Controlled by an 18-Amino-Acid C-Terminal Tail{triangledown}

Meredith T. Morris,1 Courtney DeBruin,1 Zhaoqing Yang,2 Jeremy W. Chambers,1 Kerry S. Smith,1 and James C. Morris1*

Department of Genetics and Biochemistry, Clemson University, Clemson, South Carolina 29634,1 Department of Parasitology, Kunming Medical College, Kunming, Yunnan, People's Republic of China 6500312

Received 19 May 2006/ Accepted 7 September 2006

Trypanosoma brucei expresses two hexokinases that are 98% identical, namely, TbHK1 and TbHK2. Homozygous null TbHK2–/– procyclic-form parasites exhibit an increased doubling time, a change in cell morphology, and, surprisingly, a twofold increase in cellular hexokinase activity. Recombinant TbHK1 enzymatic activity is similar to that of other hexokinases, with apparent Km values for glucose and ATP of 0.09 ± 0.02 mM and 0.28 ± 0.1 mM, respectively. The kcat value for TbHK1 is 2.9 x 104 min–1. TbHK1 can use mannose, fructose, 2-deoxyglucose, and glucosamine as substrates. In addition, TbHK1 is inhibited by fatty acids, with lauric, myristic, and palmitic acids being the most potent (with 50% inhibitory concentrations of 75.8, 78.4, and 62.4 µM, respectively). In contrast to TbHK1, recombinant TbHK2 lacks detectable enzymatic activity. Seven of the 10 amino acid differences between TbHK1 and TbHK2 lie within the C-terminal 18 amino acids of the polypeptides. Modeling of the proteins maps the C-terminal tails near the interdomain cleft of the enzyme that participates in the conformational change of the enzyme upon substrate binding. Replacing the last 18 amino acids of TbHK2 with the corresponding residues of TbHK1 yields an active recombinant protein with kinetic properties similar to those of TbHK1. Conversely, replacing the C-terminal tail of TbHK1 with the TbHK2 tail inactivates the enzyme. These findings suggest that the C-terminal tail of TbHK1 is important for hexokinase activity. The altered C-terminal tail of TbHK2, along with the phenotype of the knockout parasites, suggests a distinct function for the protein.

* Corresponding author. Mailing address: Department of Genetics and Biochemistry, Clemson University, 214 Biosystems Research Complex, 51 New Cherry Street, Clemson, SC 29634. Phone: (864) 656-0293. Fax: (864) 656-0393. E-mail: jmorri2{at}clemson.edu.

{triangledown} Published ahead of print on 6 October 2006.

Eukaryotic Cell, December 2006, p. 2014-2023, Vol. 5, No. 12
1535-9778/06/$08.00+0     doi:10.1128/EC.00146-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Chambers, J. W., Kearns, M. T., Morris, M. T., Morris, J. C. (2008). Assembly of Heterohexameric Trypanosome Hexokinases Reveals That Hexokinase 2 Is a Regulable Enzyme. J. Biol. Chem. 283: 14963-14970 [Abstract] [Full Text]  


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