With that much free space, I wouldn't think data fragmentation would become a (noticeable) problem, but it's good that using your defrag utility made a difference.
I can think of a few more tests, since you still seem to have some issues. Before you do testing on your system, it is obviously best to have a good (and recent) backup of your data.
Quit iTunes, if running. Turn OFF Wi-Fi, so that you do not have Internet access (assuming you connect to network over Wi-Fi). Launch iTunes. You'll get some messages related to not having access to iCloud. And you'll obviously have no access to the iTunes Store. Pretend you have a temporary ISP network outage (which happens in the real world), and try using iTunes for other stuff, like playing podcasts, to see if you notice a difference is slowness. When you turn Wi-Fi back ON, I would first quit iTunes, then turn Wi-Fi ON (and confirm you have an Internet connection using Safari), and launch iTunes again.
To test general Internet connection speed, you can use a web site like Speakeasy
https://www.speakeasy.net/speedtest/
I typically get 30 Mbps (download speed) or better with my service, but there are times when it is significantly slower, and that can cause iTunes to feel slower. See if there is a difference in Internet connection speed when you notice significant slowness in iTunes.
To test FireWire, most recent FireWire external drives also have a USB port. Shut down (power off) your Mac, and turn off the external drive. Disconnect the FireWire cable for the drive, and connect it with an appropriate USB cable. Turn on the external drive and start up the Mac. Run iTunes to see if you notice any difference. I'm not suggestion you don't use FireWire; this is just a test to see what happens with iTunes if your iTunes Media folder is connected over USB. Reverse procedure to put FireWire connection back.
You can also run Disk Utility (First Aid) on your startup disk. The best way is to start up the Mac through Startup Manager (hold down Option key during startup). Select Recovery HD from the list of possible startup disks. After getting to the recovery screen, select to run Disk Utility. Select your usual startup disk in Disk Utility, and run First Aid on it.