Paper describing ‘Zen-like’ approach to studying enzyme recognized as one of 2012’s best

March 06, 2013

By David Martin

Owen Nadeau and Gerald Carlson
Biochemist Gerald Carlson continues to be captivated by an enzyme he began studying in the 1970s. "It's more interesting than any person I know," he says.

Enzymes are molecules that speed up chemical reactions. The object of Carlson's interest is called phosphorylase kinase. It helps muscle cells turn glycogen into energy.

But Carlson, the chair of the Department of Biochemistry and Molecular Biology, is as intrigued by phosphorylase kinase's structure as he is by its purpose. It's large and complex, and requires what he describes as a "Zen-like" approach to studying it. "Intellectually, it's a very pleasing enzyme to work with," he says.

Carlson is not alone in his fascination. He is the senior author of a paper about phosphorylase kinase's structure that the Journal of Biological Chemistry identified as one of the best of 2012. In compiling the best of list, the editors selected 22 papers from among the more than 4,000 that were published in the journal last year.

Mysterious and elegant

Phosphorylase kinase was discovered by two American scientists, Edmond Fischer and Edwin Krebs, in the late 1950s. Fischer and Krebs were the first to describe a process for regulating proteins through their phosphorylation. This mechanism plays a role in a range of cellular activities, from the regulation of energy production to the progression of cancer. The importance of their discovery was not immediately apparent, but soon became so; Fischer and Krebs eventually won the Nobel Prize in Physiology or Medicine.

Yet phosphorylase kinase is so massive — with a molecular weight of 1.3 million — and complicated that it has remained something of a mystery, particularly its structure. It has four copies of four subunits, or component proteins, which always remain firmly bound together within the enzyme complex. Carlson's group hoped to learn the structure of a specific subunit within the complex while in the presence of all the others. A process, he stated, "that required diverse and creative approaches."

He adds: "I think of our enzyme as a mysterious and elegant woman from probably the 18th or 19th century that you must very delicately and indirectly question and then patiently wait to see what she elects to share with you."

A trip to Oxford

An earlier Carlson-led study had determined that phosphorylase kinase was composed of two equivalent lobes connected by four bridges. For the current project, Carlson and his team set out to determine the structure of the "beta" subunits within the complex. The beta subunit is phosphorylated in response to adrenalin, playing a key role in the fight-or-flight response in mammals, and makes up almost 40 percent of phosphorylase kinase's mass.

The enzyme's enigmatic nature required a combination of chemical, biochemical, biophysical and computational techniques. The Carlson lab teased out so much data that the researchers designed a search engine to make sense of it all. "The amount of information when you try to perform this kind of study is enormous," says Owen Nadeau, research assistant professor of biochemistry and molecular biology and the paper's lead author.

At one point in the project, Nadeau traveled to the University of Oxford. He spent two weeks in England collaborating with chemists who created a mass spectrometer capable of handling an enzyme of phosphorylase kinase's heft.

The effort paid off. Carlson, Nadeau and their colleagues were able to learn a lot of fine structural details about the beta subunit while in the presence of all the other subunits that make up the enzyme complex. The study confirmed an earlier Carlson hypothesis that the four beta subunits of phosphorylase kinase compose the complex's four bridges and form the enzyme's structural core.

Those who aren't protein chemists or structural biologists may struggle to comprehend the research. But anyone can appreciate the dedication required to dissect the subunit's structure. The paper was four years in the making.

"It was mostly just determination to learn all we could about the beta subunit." Carlson says. "We just keep figuring out new ways to attack it, and they all kind of fell together."

Co-authors of the study from the University of Kansas Medical Center include Antonio Artigues, research associate professor of biochemistry and molecular biology and director of the mass spectrometry/proteomics laboratory, his assistant Maria Villar, and Jessica Sage, Timothy Priddy and Qing Yang from the Carlson laboratory. In addition to the University of Oxford, the KUMC researchers also collaborated with colleagues at the University of Michigan. The study was supported by grant R01 DK32953 from the National Institutes of Health.
Last modified: Apr 28, 2014