Henry Jakubowski

Chemistry Department

College of St. Benedict/St. John's University

Professor of Chemistry, 2002

Ph.D. University of Iowa, 1986

B.S. - State University of New York at Albany, 1975

Email:  hjakubowski@csbsju.edu

Phone:  320-363-5354

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Mutagenesis of Human Adipocyte Acid Phospatase Beta (HAAP-b) or

Low Molecular Weight Protein Tyrosine Phosphatase (LMW PTP)

Enzymes that cleave phosphate groups from proteins are abundant in all cells.  These enzymes, called phosphatases, regulate the activity of phosphorylated proteins, rendering the dephosphorylated proteins either active or inactive.  Phosphatases are key proteins in the regulation of cells growth and metabolic state. I am studying human adipocyte acid phosphatase (found in human fat cells).  Dr. McIntee and I are in the process of writing a large grant to the NIH to study the natural protein substrates of this enzyme and to develop drugs to inhibit the activity of the enzyme. 

We actually let bacteria make HAAP-b for us.  The human gene for the protein is placed in E. Coli and induced to express the protein, a cartoon of which in the figure below.  The active site of the enzyme, where the chemical cleavage of the phosphate group occurs,  must bind phosphate groups which are covalently attached to proteins.  The activity of the enzyme can be monitored easily in solution using p-nitrophenol phosphate which is cleaved by the enzyme to produce a yellow solution.  An active site cysteine (Cys 12) acts as a nucleophile in the cleavage reaction.  The enzyme binds phosphate in the active site, which inhibits the enzyme.

My research involves making mutations in the DNA for HAAP-b and studying the effect of these mutants on HAAP-b activity.  We need to make at least three mutation.  My student last year, Claire Hoolihan, made one of them.  The mutants can be divided into two groups:

1.      Active site mutant:  We wish to mutate the DNA for the gene so that in the mutant protein, the active site Cys (C) is replaced for a Ser (S) at amino acid 12 (C12S).   This makes the enzyme catalytically inactive, unable to cleave phosphate groups from proteins. However, this mutant will still be able to bind phospho-proteins.  We will use this mutant in future research to bind to natural phospho-proteins in epithelial and fat cells, and to identify the binding sites on those phospho-proteins for HAAP-b.

2.      Nonactive site mutant:  HAAP-b contains two tryptophans (W).  We will make two different mutants, changing a single W to phenyalanine (F) in each one, thereby producing two mutants that contain only one W residue.  We have already made one mutation, changing W49, an amino acid that is near the active site and which fluoresces, to F which does not fluoresce.  We will make a second mutant to change W39, located on the opposite side from the active site, to a phenylalanine.  Fluorescence from the mutant with only a single W at position 49 will be sensitive to the environment of the active site, while the other mutant, containing a single W at position 39, will be used to detect changes in protein structure away from the active site, and near an interesting positively-charged N terminus (shown as a red loop in the cartoon below) which is not found in other homologous acid phosphatases.

The mutants will be made, expressed in E. Coli, and purified.  The activity of the two mutant proteins, as measured by cleavage of p-nitrophenyl phosphate and fluorescence, in the presence and absence of different inhibitors of the enzyme, will be used to better understand how the structure of the enzyme influences its activity.  In addition, the stability and unfolding of the protein will be studied used fluorescence from the single tryptophan-containing mutants. 

We will also make a double mutant:  C12S and 39WF, producing a protein that can not cleave phosphates from target proteins (C12S) and which has a single W at position 49 (W39F) which will be used to monitor phospho-protein binding to the double mutant by monitoring fluorescence changes in W49. 

After the mutants are made, they will be purified and their kinetic activity against p-nitrophenylphosphate monitored. 

Applications of Fluorescence Measurements in Biochemistry

My second project involves using the spectrofluorometer to study a range of biological questions.  When molecules absorb UV or visible light, electrons are excited to higher energy levels.  The excited electrons can “relax” back to lower energy levels by losing energy through collisions or by emitting photons of lower energy than the original excitation photons.  This emission is called fluorescence.  In contrast to simple absorbance properties, fluorescence emission is extremely sensitive to the environment of the flourophore, the molecule that emits.  The properties of biological molecules can be studied using fluorescence.   Two types of fluorophores are used.  Intrinsic fluorophores are part of the actual molecule (for example a tryptophan side chain in a protein).  Extrinsic fluorophores (like fluorescein) can be attached (covalently or noncovalently) to proteins, lipid aggregates and DNA.   

Students in BCHM 321 conduct independent research projects using fluorescence to study proteins, lipids, and DNA.   This project involves developing new applications of fluorescence spectroscopy to study the structure/function of biological molecules, including lipids, proteins, and nucleic acids

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