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Contribution of amino acid region 334-335 from factor Va heavy chain to the catalytic efficiency of prothrombinase.

ABSTRACT: We have demonstrated that amino acids E (323), Y (324), E (330), and V (331) from the factor Va heavy chain are required for the interaction of the cofactor with factor Xa and optimum rates of prothrombin cleavage. We have also shown that amino acid region 332-336 contains residues that are important for cofactor function. Using overlapping peptides, we identified amino acids D (334) and Y (335) as contributors to cofactor activity. We constructed recombinant factor V molecules with the mutations D (334) --> K and Y (335) --> F (factor V (KF)) and D (334) --> A and Y (335) --> A (factor V (AA)). Kinetic studies showed that while factor Va (KF) and factor Va (AA) had a K D for factor Xa similar to the K D observed for wild-type factor Va (factor Va (WT)), the clotting activities of the mutant molecules were impaired and the k cat of prothrombinase assembled with factor Va (KF) and factor Va (AA) was reduced. The second-order rate constant of prothrombinase assembled with factor Va (KF) or factor Va (AA) for prothrombin activation was approximately 10-fold lower than the second-order rate constant for the same reaction catalyzed by prothrombinase assembled with factor Va (WT). We also created quadruple mutants combining mutations in the amino acid region 334-335 with mutations at the previously identified amino acids that are important for factor Xa binding (i.e., E (323)Y (324) and E (330)V (331)). Prothrombinase assembled with the quadruple mutant molecules displayed a second-order rate constant up to 400-fold lower than the values obtained with prothrombinase assembled with factor Va (WT). The data demonstrate that amino acid region 334-335 is required for the rearrangement of enzyme and substrate necessary for efficient catalysis of prothrombin by prothrombinase.

SUBMITTER: Barhoover MA

PROVIDER: S-EPMC2652359 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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Contribution of amino acid region 334-335 from factor Va heavy chain to the catalytic efficiency of prothrombinase.

Barhoover Melissa A MA Orban Tivadar T Beck Daniel O DO Bukys Michael A MA Kalafatis Michael M

Biochemistry 20080607 26

We have demonstrated that amino acids E (323), Y (324), E (330), and V (331) from the factor Va heavy chain are required for the interaction of the cofactor with factor Xa and optimum rates of prothrombin cleavage. We have also shown that amino acid region 332-336 contains residues that are important for cofactor function. Using overlapping peptides, we identified amino acids D (334) and Y (335) as contributors to cofactor activity. We constructed recombinant factor V molecules with the mutation ...[more]

PMID: 18537263

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Project description:Prediction of protein secondary structure from FTIR spectra usually relies on the absorbance in the amide I-amide II region of the spectrum. It assumes that the absorbance in this spectral region, i.e., roughly 1700-1500 cm-1 is solely arising from amide contributions. Yet, it is accepted that, on the average, about 20% of the absorbance is due to amino acid side chains. The present paper evaluates the contribution of amino acid side chains in this spectral region and the potential to improve secondary structure prediction after correcting for their contribution. We show that the β-sheet content prediction is improved upon subtraction of amino acid side chain contributions in the amide I-amide II spectral range. Improvement is relatively important, for instance, the error of prediction of β-sheet content decreases from 5.42 to 4.97% when evaluated by ascending stepwise regression. Other methods tested such as partial least square regression and support vector machine have also improved accuracy for β-sheet content evaluation. The other structures such as α-helix do not significantly benefit from side chain contribution subtraction, in some cases prediction is even degraded. We show that co-linearity between secondary structure content and amino acid composition is not a main limitation for improving secondary structure prediction. We also show that, even though based on different criteria, secondary structures defined by DSSP and XTLSSTR both arrive at the same conclusion: only the β-sheet structure clearly benefits from side chain subtraction. It must be concluded that side chain contribution subtraction benefit for the evaluation of other secondary structure contents is limited by the very rough description of side chain absorbance which does not take into account the variations related to their environment. The study was performed on a large protein set. To deal with the large number of proteins present, we worked on protein microarrays deposited on BaF2 slides and FTIR spectra were acquired with an imaging system.

Dataset Information

Contribution of amino acid region 334-335 from factor Va heavy chain to the catalytic efficiency of prothrombinase.

Publications

Contribution of amino acid region 334-335 from factor Va heavy chain to the catalytic efficiency of prothrombinase.

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